Pmi127 LM

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PMI 127
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Laboratory Manual
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Spring 2015
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Editors: Lisa Tran
Rance LeFebvre
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Course Introduction
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Welcome to PMI 127, Medical Microbiology. In this class, you will be working with
many pathogens isolated from samples submitted to the UCD VetMed Teaching Hospital’s
microbiology lab. Some of these pathogens may have been the cause of death in many of the
animals so special care must be taken with any live culture you will encounter.
When working up the cultures, never leave plate media or biochemical tests in the
incubator for more than 24 hours unless otherwise specified. The lab protocols will state
when a test must be read within 24 hours. Leaving the plate media in the incubator will cause it
to dehydrate and crack. Lab assistants will remove your cultures from the incubator after 24
hours and place them in the refrigerator but it will be your responsibility to interpret your results
in a timely manner. All other types of media (broths, tubes, biochemical tests) can and should be
incubated for over 24 hours for best results.
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During lab:
- Be sure to read the notes at the bottom of the lab exercises, they will often contain
important reminders about the day’s cultures
- Always label your cultures with your name, date, organism and group number. You may
also find it helpful to mark your plates and tubes to make them easier to identify.
- NEVER leave plate media and biochemical tests in the incubator for more then 24 hours
unless otherwise specified.
- Check the lab manual for solutions to any questions you may have before going to the
TA’s.
- Unannounced lab quizzes may be given. They can be worth up to 5 points and are good
study guides for the lab practical.
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Your responsibilities in this class:
- Read and be familiar with the day’s protocol and background information. The
introductions include detailed information and methods on tests and biochemical media
you will be using.
- Practice safe and aseptic laboratory techniques. That includes disinfecting your work area
before and after lab. We will be working with live cultures and open flames. Personal
protective equipment will be provided upon request.
- Maintain overall lab cleanliness. The TA’s are here to help with course related questions,
not to clean up after the students.
- Come in during non-lab days to record Unknown results.
Unknown write-ups:
- You are responsible for coming in on non-lab days (except weekends) to work up your
unknowns, record your results and remove your media from the incubator.
- Write-ups are due one week after they are assigned
- A complete report includes three parts:
o Summary sheet, all parts filled out, complete with date and result of each
diagnostic test performed
o Typed or neatly written explanation of your procedures and results
o Flowchart of all infectious possibilities and diagnostic tests needed to identify
each one.
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Tables
Tables should be filled out but will not be collected or graded. These tables are for your
reference only and will be helpful to have for your unknowns and lab practical.
The laboratory exercises are designed to introduce and familiarize you with the
approaches and techniques for diagnosis of bacterial and fungal diseases. Knowledge of
diagnostic methods available will allow you to confidently select tests for diagnoses of clinical
disease. This will apply whether the tests are performed within your practice or by an outside
diagnostic laboratory.
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Objectives of Laboratory Sessions
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1. To learn to develop a strategy for disease diagnosis.
2. To gain proficiency in diagnostic methods that are applicable in a basic clinical
laboratory.
3. To develop skills in sterile technique.
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The basis of the laboratory exercises is a limited battery of culture media, biochemical tests,
and equipment that reflects the general requirements for a reasonable Diagnostic Bacteriology
Laboratory in a private veterinary practice. This diagnostic battery will be utilized throughout the
laboratory exercises. Additional tests available at referral laboratories such as the Veterinary
Medicine Teaching Hospital (VMTH) will be introduced during the course.
Laboratory Rules
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As a Microbiologist you will be responsible for ensuring that specimen collection,
handling, and testing is conducted properly and without health risk to you or your practice staff.
In order to direct these procedures in a practice, you must become familiar with and dedicated to
basic laboratory safety.
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The safety of everyone in the laboratory depends upon strict observation of these rules.
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1. Treat all cultures as potential human pathogens, including those isolated from your skin
and around the classroom.
2. This laboratory is designated as a Biosafety Level 2 lab, which means there is no food or
drinks or pets allowed in the lab. All food must be left outside the classroom.
3. Proper aseptic technique must always be practiced. This includes proper hand washing,
sterilization of all work surfaces before and after use and disposing of class materials in
the proper waste containers.
a. Wash your hands with soap before each lab to prevent introducing potential
pathogens into class cultures and after each lab to prevent carrying potential
pathogens outside the classroom.
b. Wipe down work spaces including bench tops and sinks with 70% EtOH before
and after class.
4. Avoid having unnecessary objects cluttering your work space. This reduces the risk of
cross contamination.
5. Dispose of all cultures in the red, plastic, biohazard buckets that have been labeled with
biohazard stickers.
6. Dispose of all microscope slides and broken glass in the sharps bucket. These are the red,
plastic boxes with white lids located near the sinks.
7. Long hair must be tied back and loose pieces of clothing must be secured to avoid being
burned by the Bunsen burner.
8. Any spills or accidents must be reported to the lab instructor or the teaching assistants.
Laboratory Procedures
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1. All cultures will be provided on one of three forms of media: broths, slants, or plates.
2. Unless otherwise specified, all cultures will be incubated aerobically at 37ºC for 24
hours.
3. Label all cultures with your name, the date, and the organism name, number or source.
4. Dispose of all used media in the red, plastic, double lined biohazard buckets located
around the lab. Dispose of all slides and broken glass in the red, plastic sharps containers
located at each sink station. Dispose of all non-contaminated waste in trash cans located
around the lab.
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Accidents
1. In case of spills,
a. Notify others of the spill so the contamination does not spread.
b. Cover the spill area with disinfectant and lay a paper towel on top of the spill and
soak the paper towel with more disinfectant.
c. Notify the instructor of the spill.
2. In case of injury, immediately notify the instructor and fill out an accident report form.
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Fire
1. Lab fire: Turn off the gas and put out the fire. Notify the instructor.
2. Alarm: Calmly leave by the nearest door and exit by the staircase. Meet at the grassy area in
front of the building.
Experiments
Experiment*# Title* pages
1 Meet$the$Staph:$Identification$of$Staphylococci 1.1$5$1.4
2 Staph$only,$please:$Isolation$of!S.!aureus$from$self 2.1$5$2.2
3 Identification$of$Streptococci 3.1$5$3.5
4 Identification$of$Bordetella 4.1$5$4.3
5 Identification$of$Corynebacterium 5.1$5$5.2
6 Identfication$of$Haemophilus!influenzae 6.1$5$6.3
7 Identification$of$Enterobacteriaceae 7.1$5$7.5
8 SNL TBD
9 That$was$easy:$EnteroPluri$test 9.1
10 Got$food$poisoning?$Could$B.!cereus. 10.1$5$10.2
11 Antibiotic$sensitivity$testing 11.1$5$11.3
12 Fungus$is$among$us:$Identification$of$yeasts 12.1$5$12.2
Experiment*1:*Meet*the*Staph*
Goal:&Identify&and&differentiate&between&pathogenic&and&non6pathogenic&Staphylococci.&
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Staphylococcus&epidermidis!is!a!normal!inhabitant!of!the!human!skin.!Although&S.&epidermidis!
has!not!evolved!to!cause!disease,!it!is!known!for!its!involvement!in!the!production!of!biofilms!
on!hospital!apparati!used!in!invasive!procedures!(Michal!Otto,!2009).!Staphylococcus&aureus!is!
another!normal!inhabitant!of!the!human!skin.!Unlike!S.&epidermidis,!S.&aureus!has!evolved!to!
cause!disease.!In!this!lab,!you!will!run!several!biochemical!tests!to!differentiate!between!the!2!
Staphylococcus!species.!Micrococcus!is!found!on!dust,!on!the!skin,!in!the!air,!etc.!It!is!included!
for!comparative!purposes.!
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Day*1*
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Materials:!(per!group)!
$ 1!tube!of!mixed!culture!(contains!S.&aureus,&S.&epidermidis!and!Micrococcus)!
$ 1!SBA!plate!
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Procedure:!
$ Direct!smear!of!mixed!culture.!
o Direct!smears!are!usually!Gram!stains!of!the!raw!sample.!A!direct!smear!will!usually!
contain!cellular!debris!and!normal!flora!associated!with!the!site!at!which!the!sample!
was!taken.!When!properly!read,!direct!smears!can!narrow!down,!or!even!confirm,!
the!identity!of!your!suspect!by!providing!valuable!information!including,!but!not!
limited!to:!
1. Replicative!morphology!
2. Ability!to!live!intracellularly!
3. Arrangement!of!cells!in!tissue!
o In!this!class,!you!will!be!performing!Gram!stains!as!your!direct!smears.!Remember!
that!a!Gram!stain!can!only!be!called!a!direct!smear!if!the!stain!is!of!the!raw!sample,!
not!a!colony!taken!from!culturing!media.!
$ Streak!contents!of!the!tube!for!isolation!onto!SBA!
o Streaking!a!sample!for!isolation!is!a!crucial!technique!in!identifying!bacteria.!The!
sample!is!first!inoculated!into!the!first!quadrant.!Then!a!flameQsterilized,!cooled!
loop!is!used!to!streak!the!remaining!quadrants.!The!previous!quadrant!is!always!the!
one!used!to!inoculate!the!next!quadrant!and!the!loop!is!always!flameQsterilized!and!
cooled!between!quadrants.!For!stepQbyQstep!instructions,!see!below:!
1. FlameQsterilize!and!cool!loop.!!
2. Pick!up!a!loopful!of!sample!and!spread!it!evenly!over!quadrant!1.!
3. FlameQsterilize!and!cool!loop.!!
4. Streak!quadrant!2,!making!sure!to!overlap!quadrant!1!no!more!than!3!times.!
5. Repeat!steps!3!and!4!for!the!last!quadrant.!!
6. FlameQsterilize!loop.!
1.1!
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o The!idea!behind!streaking!3!quadrants!is!to!dilute!the!inoculum!with!every!
subsequent!quadrant.!Flaming!the!loop!in!between!each!quadrant!will!remove!any!
excess!bacteria!from!the!loop.!The!bacterial!numbers!will!decrease!with!every!
quadrant!until!we!have!isolated!colonies!(Figure!1.1).!!
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Figure*1.1:*Adopted*from*ASM*MicrobeLibrary.*The*image*on*the*left*depicts*a*wellBstreaked*plate*with*many*
isolated*colonies.*The*plate*on*the*right*did*not*achieve*isolation.*This*may*be*due*to*several*factors:*1)*the*
first*quadrant*should*not*be*overlapped*more*than*3*times*and*2)*the*loop*should*be*sterilized*in*between*
each*quadrant*to*remove*excess*bacteria.**
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Day*2*
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$ 2!SBA!plates!
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Procedure:!
$ Examine!plates!for!growth,!making!note!of!unique!colony!morphologies.!!
o In!addition!to!Gram!stains,!observation!of!colony!morphology!is!another!powerful!
tool!in!specimen!identification.!Many!bacteria!have!distinguishing!colony!
morphologies!that!help!to!point!you!in!the!right!direction.!!
$ Pick!wellQisolated!colonies!with!unique!morphologies!to!subculture!onto!new!plates.!
o Picking!wellQisolated!colonies!for!pure!culture!is!the!crucial!first!step!to!successfully!
identifying!your!samples.!You!will!often!be!asked!to!pick!a!colony!from!selective!
media!to!subculture!onto!SBA!plates.!Although!colonies!may!be!clearly!
distinguishable!on!selective!agar,!many!bacteria!appear!the!same!on!SBA.!!
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Day*3*
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$ 2!coagulase!tests!
$ 1!mannitol!salt!agar!plate!
1.2!
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Procedure:!
$ Examine!plates!for!growth.!You!should!have!pure!cultures!of!each!unique!colony.!!!
o Note!colony!color,!size,!dimensions,!hemolysis,!etc.!Describe!morphologies!as!
specifically!as!possible.!This!will!make!your!work!easier!throughout!the!quarter!
when!identifying!other!“mediumQsized,!round,!smooth”!colonies.!It!may!even!help!
to!diagram!the!colonies!in!your!notes.!
$ Perform!catalase!test!on!each!sample.!
o The!catalase!test!is!a!rapid!biochemical!test!that!is!used!to!test!for!the!production!of!
the!enzyme!catalase.!It!is!most!commonly!used!to!differentiate!between!the!genera!
Staphylococcus!and!Streptococcus.!Catalase!converts!H2O2!into!water!and!gaseous!
oxygen.!To!perform!the!catalase!test,!put!a!loopful!of!the!isolate!onto!a!clean!glass!
slide.!Add!a!drop!of!catalase!reagent!(hydrogen!peroxide).!When!H2O2!(hydrogen!
peroxide)!is!added!onto!a!catalaseQpositive!culture,!oxygen!bubbles!will!form!
immediately.!No!bubbles!will!form!in!the!presence!of!a!catalaseQnegative!culture!
(Figure!1.2).!!
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Figure*1.2:*Adopted*from*ASM*MicrobeLibrary.*Top:*Positive*reaction*from*Staphylococcus,aureus.*Bottom:*
negative*reaction*from*Streptococcus,pyogenes.*
$ Inoculate!one!coagulase!test!for!each!sample.!!
o The!coagulase!test!is!used!to!test!for!the!production!of!the!enzyme!coagulase.!It!is!
used!to!differentiate!Staphylococcus&aureus!from!other!GramQpositive!cocci.!
Coagulase!works!in!conjunction!with!normal!plasma!components!to!form!protective!
fibrin!barriers!around!individual!cells!or!groups!of!cells.!The!test!is!carried!out!by!
inoculating!a!tube!of!rabbit!serum!with!a!colony!from!a!pure!culture.!Tests!are!read!
4B24*hours!after!inoculation.!A!positive!result!is!characterized!by!coagulation!of!the!
rabbit!serum!while!there!will!be!no!change!in!viscosity!in!the!negative!tube.!Only!
pathogenic!Staphylococci!are!able!to!produce!a!positive!result.!!
$ Divide!a!mannitol!salt!plate!in!half!and!streak!one!half!with!a!colony!from!one!plate!and!the!
other!half!with!a!colony!from!the!second!plate.!!
o Mannitol!salt!agar!is!a!selective!and!differential!medium!used!to!differentiate!S.&
aureus!from!S.&epidermidis.!Mannitol!salt!agar!is!high!in!salt!concentration,!
prohibiting!the!growth!of!many!other!bacteria.!Mannitol!is!used!to!differentiate!
between!organisms!that!can!ferment!mannitol!from!organisms!that!cannot.!
Fermentation!of!the!mannitol!will!turn!the!pH!indicator!in!the!agar!a!bright!yellow!
due!to!the!change!in!pH.!!
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1.3!
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Day*4*
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$ No!additional!materials!needed.!
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Procedure:!
$ Examine!and!record!results!in!the!table!below.!!
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Experiment*1:*Meet*the*Staph**
!! S.,aureus, S.,epidermidis, Micrococcus,
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Colony*morphology*(on*
SBA)*Note*colony*color,*
size,*dimensions,*margin,*
etc.*
!! !! !!
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Hemolysis* !! !! !!
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Gram*stain*(note*GramB
ness,*cell*morphology,*as*
well*as*cell*arrangement)*
!! ! !!
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Catalase* !! !!
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Mannitol*fermentation* !! !! Q!
Coagulase* !! !! Q!
7.5%*NaCl* +! +! +!
DNAase* +! Q! Q!
ThermalBstable*DNAase* +! Q! Q!
1.4!
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Experiment*2:*Staph*only,*please.*
Goal:&Isolation&of&Staphylococcus&aureus&from&self&(nasal&or&throat&culture)&&
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Day*1*
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Materials:!(per!person)!
% 1!sterile,!cotton3tipped!applicator!
% 1!SBA!plate!
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Procedure:!
% Swab!anterior!nares!or!tonsils.!!
% Inoculate!the!first!quadrant!of!the!SBA!plate!with!the!swab!and!streak!the!remainder!of!the!
plate!for!isolation.!
% Direct!smear!of!the!swab.!!
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Day*2*
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% 1!SBA!plate!
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Procedure:!
% Gram!stain!unique!colonies.!
% Streak!S.&aureus!suspects!for!isolation.!Suspects!must!be!β3hemolytic.!
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Day*3*
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% 1!mannitol!salt!agar!plate!
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Procedure:!
% Make!sure!your!S.&aureus!suspect!is:!
o A!Gram3positive!coccus!
o Catalase!positive!
o β3hemolytic!
% If!your!suspect!meets!the!3!criteria!above,!inoculate!a!mannitol!salt!agar!plate!and/or!a!
coagulase!test!to!determine!if!it!is!S.&aureus.!
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Day*4*
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% 1!Nutrient!agar!slant!
! 2.1$
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Procedure:!
% If!the!suspect!meets!all!requirements,!streak!a!nutrient!agar!slant!from!a!colony!on!the!
mannitol!salt!agar!plate.!!
% Make!sure!you!clearly!label!the!slant!with!your!name!and!group!#.!Give!your!slant!to!the!
TA.!The!slants!will!be!incubated!overnight!and!saved!for!future!use.!!
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Experiment*2:*Nasal*Culture*results*
! Suspect*colony* *
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Sample*site*(Nose*or*throat?)*
Colony*morphology*(on*SBA)*
(Note*colony*color,*size,*
dimensions,*etc.)*
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Hemolysis* !
Gram*stain*
(Note*GramHness,*cell*
morphology,*as*well*as*cell*
arrangement.)*
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Catalase* !
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Mannitol*fermentation* !
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Coagulase**
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Identity*of*suspect*organism* !
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! 2.2$
Experiment*3:*Identification*of*Streptococci*
Goal:&Identify&and&differentiate&between&various&pathogenic&and&non9pathogenic&Streptococci.&&
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Streptococci!form!colonies!that!are!distinctly!smaller!than!those!of!staphylococci.!Due!to!the!
small!size!of!the!colonies,!we!will!rely!heavily!on!the!degree!of!hemolysis!to!differentiate!
between!different!species.!In!this!lab,!you!will!differentiate!between!4!species!of!Streptococcus!
and!one!species!of!Enterococcus.!As!you!go!through!the!lab,!be!sure!to!compare!and!contrast!
these!results!to!those!of!Staphylococcus.!!
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Day*1*
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% 5!tubes!of!broth!labeled!ADE!(tubes!contain!pure!cultures!of!S.&pyogenes,&S.&agalactiae,&S.&
pneumoniae,&S.&viridans!or!Enterococcus)!
% 5!SBA!plates!
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Procedure:!
% Direct!smear!of!each!sample.!
% Streak!the!contents!of!each!tube!for!isolation!on!SBA.!!
% Please*SAVE*the*broth*cultures*at*4ºC*for*the*next*lab*period!*
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Day*2*
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% 5!broth!cultures!from!day!1!
% 3!SBA!plates!
% 2!optochin!disks!
% 2!bacitracin!disks!
% 4!sterile!cotton!applicators!
% S.&aureus!on!SBA!!
% 1!Bile!Esculin!Agar!plate!
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Procedure:!
% Examine!plates!for!growth.!Record!colony!morphologies.!
o When!looking!for!different!types!of!colonies,!it!is!important!to!look!at!the!effect!of!
each!colony!on!the!red!blood!cells!in!the!agar.!Streptococci!only!develop!into!small,!
pinpoint!colonies!after!overnight!incubation,!so!we!will!rely!heavily!on!the!different!
degrees!of!hemolysis!during!this!stage!of!identification.!
o Hemolysis!is!the!lysis!of!red!blood!cells.!It!is!very!useful!in!the!identification!of!
streptococci.!For!Streptococcus!and!ONLY!Streptococcus,!their!effect!on!blood!can!
be!classified!as!αDhemolysis,!βDhemolysis,!or!γ!hemolysis.!!
3.1$
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! αDhemolysis!appears!as!a!greenish!zone!under!and!around!each!colony.!
Bacterial!products!have!altered!the!hemoglobin!in!the!RBCs!but!the!RBCs!
have!NOT!been!lysed.!
! βDhemolysis!appears!as!a!zone!of!clearing!under!and!around!each!colony.!
The!RBCs!have!been!lysed!and!affected!portions!of!the!agar!appear!
transparent.!!
! γDhemolysis!has!no!effect!on!the!agar!and!the!RBCs!remain!intact.!!
o Blood!agar!that!has!been!incubated!for!too!long!or!at!too!high!a!temperature!will!
begin!to!turn!a!greenishDbrown!and!is!often!misinterpreted!as!being!αDhemolysis.!
For!there!to!be!true!hemolysis,!the!RBCs!under!and!around!each!colony!must!be!
affected,!not!just!the!ones!around!the!colonies!in!quadrant!1.!!
o Other!types!of!bacteria!can!also!be!hemolytic,!but!their!hemolysis!is!not!
categorized.!They!are!recognized!as!either!hemolytic!(as!in!βDhemolytic)!or!nonD
hemolytic.!!
% Perform!a!catalase!test!on!each!sample.!!
% Set!up!optochin!test!with!αDhemolytic!isolates.!
o Optochin!is!an!antibiotic!used!to!differentiate!between!S.&pneumoniae!and!S.&
viridans.!To!set!up!the!test,!divide!a!SBA!plate!in!half!(Figure!3.1a).!Using!a!sterile!
cotton!applicator,!paint!one!half!of!the!plate!with!an!even!lawn!of!one!αDhemolytic!
broth!culture!(Figure!3.1b).!Repeat!this!step!with!the!other!αDhemolytic!culture!on!
the!other!half!of!the!plate.!Once!the!lawns!are!painted,!use!sterile!forceps!to!place!1!
optochin!disk!in!the!center!of!each!lawn!(Figure!3.1c).!!!
!
!! !!!!!!!!!!!Figure!3.1a! ! !!!!!!!Figure!3.1b! ! !!!!!Figure!3.1c!
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% Set!up!bacitracin!test!with!βDhemolytic!isolates.!
o Bacitracin!is!an!antibiotic!used!to!differentiate!between!S.&pyogenes!and!S.&
agalactiae.!Set!up!the!test!as!detailed!above.!!
% Set!up!CAMP!test!with!βDhemolytic!samples.!
o CAMP!is!an!acronym!for!the!developers!of!the!test:!Christie,!Atkins!and!MunchD
Petersen.!It!is!used!to!differentiate!between!S.&agalactiae!and!other!Streptococcus!
species.!Group!B!Strep!(S.&agalactiae)!produces!a!hemolytic!protein!that!works!
synergistically!with!the!βDhemolysin!of!S.&aureus.!When!streaked!perpendicular!to!a!
S.&aureus!streak!on!SBA,!S.&agalactiae!will!produce!an!arrowheadDshaped!enhanced!
zone!of!hemolysis.!!
3.2$
!
o To!set!up!this!test!(Figure!3.2a),!first!streak!a!line!of!S.&aureus!down!the!center!of!a!
SBA!plate!(labeled!as!“1).!Then!streak!the!suspect!organism(s)!perpendicular!to!the!
S.&aureus!streak!(labeled!as!“2”!and!“3”).!Start!from!the!edge!of!the!plate!and!streak!
toward!the!center,!as!indicated!by!the!directional!arrow.!The!expected!results!from!
the!test!are!shown!in!Figure!3.2b.!GBS!will!have!an!enhanced!zone!of!hemolysis.!
! ! !
!! ! !!Figure!3.2a! ! ! ! ! !!!!!!!!Figure!3.2b!
!
% Inoculate!a!Bile!Esculin!Agar!plate!with!all!suspects.!
o Bile!Esculin!Agar!is!a!selective!and!differential!medium!containing!bile,!esculin!and!
ferric!ammonium!citrate.!Bile!serves!as!a!selective!agent,!allowing!for!the!growth!of!
enterococci!while!inhibiting!the!growth!of!other!streptococci.!This!agar!is!used!to!
test!for!the!hydrolysis!of!esculin.!When!esculin!molecules!are!split,!the!resulting!
esculetin!reacts!with!iron!(from!ferric!ammonium!citrate)!to!form!a!dark!brown!
precipitate.!
o To!inoculate!this!test,!see!figure!3.2.!First,!mark!5!parallel!lines!on!the!bottom!of!the!
petri!plate!and!label!them!with!letters!ADE.!Inoculate!the!plate!with!a!loopful!of!each!
culture!on!the!corresponding!line.!There!is!no!need!to!streak!for!isolation.!We!are!
testing!solely!for!the!presence!or!absence!of!growth!and!a!dark!brown!precipitate.!
! ! !
!! ! !Figure!3.2! ! ! ! ! !!Expected!Results!
*
*
3.3$
!
Day*3*
!
% none!
!
Procedure:!
% Examine!antibiotic!testing!plates.!Record!results.!!
% Examine!CAMP!test!results.!
% Examine!Bile!Esculin!Agar.!Record!results.!
!
!
Results:*
* A* B* C* D* E*
!
Gram*stain*
!
(GramHness,*cell* ! ! ! !
!
morphology,*etc.)*
!
!
Colony*
!
morphology* ! ! ! !
!
(color,*size,*etc.)*
!
!
Hemolysis**
! ! ! ! !
(α,*β,*or*γ)!
!
!
Catalase* ! ! ! !
!
!
Bile*Esculin*agar* ! ! ! !
!
!
Suspect*organism* ! ! ! ! !
!
!
** ******Optochin* * * **********Bacitracin* * * ******CAMP*
$ $ $
3.4$
!
Experiment%3:%Identification%of%Streptococci
Enterococcus*1 S.*pyogenes S.*agalactiae S.*pneumoniae S.*viridans
Gram%stain%(note%
Gram-ness%as%well%as%
cell%morphology%and%
cell%arrangement)
Colony%morphology%
(on%SBA)%Note%colony%
color,%size,%
dimensions,%margin,%
etc.
Hemolysis%(α,%β,%or%γ)
Catalase
Lancefield%Group%1
CAMP%test% –– –– ––
Bacitracin%2 –– –– ––
Optochin%2 –– –– ––
Bile%esculin%3
6.5%%NaCl + - - - -
Lactose d + + + d
Sorbitol d - - d -
Trehalolse d + + d +
Soluble%hemoysin - + d - -
Hippurate –– - + –– ––
1"
Lancefield"Grouping:"see"Appendix
2"
Note"results"as"sensitive"or"resistant."It"may"even"help"to"note"the"size"of"the"clear"zone."
3
"Note"the"presence"of"growth"as"well"as"the"color"of"the"colonies.
Experiment*4:*Identification*of*Bordetella)pertussis**
*
*
Day*1*
*
% 1!tube!of!pure!culture!of!Bordetella)pertussis!
% 1!MacConkey!agar!
% 1!SBA!plate!
!
Procedure:!
% Direct!smear.!
% Streak!contents!of!the!tube!for!isolation!onto!MacConkey!agar.!
o MacConkey!agar!is!a!selective!medium!used!primarily!for!the!isolation!and!
purification!of!enterics.!There!are,!however,!a!few!nonFenterics!that!can!grow!on!
MacConkey!agar,!two!of!which!will!be!covered!in!the!scope!of!this!course.!These!
two!genera!are!Bordetella!and!Pseudomonas.!MacConkey!agar!contains!bile!salts!
and!crystal!violet,!both!of!which!inhibit!the!growth!of!GramFpositive!bacteria.!!
% Streak!contents!of!the!tube!for!isolation!onto!SBA.!
*
*
Day*2*
!
% Oxidase!
% 3%!KOH!
% 1!ReganFLowe!Charcoal!agar!
!
Procedure:!
% Perform!oxidase!test!on!Bordetella.!
o Purpose:!The!oxidase!test!is!a!rapid!biochemical!test!used!to!differentiate!between!
the!oxidaseFpositive!Bordetella)and)Pseudomonas!from!the!oxidaseFnegative!
Enterobacteriaceae.!!
o How!it!works:!It!detects!for!the!presence!of!a!respiratory!enzyme!called!cytochrome!
c!oxidase.!Members!of!Enterobacteriaceae!have!a!different!terminal!oxidase!system!
and!will!therefore!give!a!negative!result.!!
o To!run!the!test:!
! Smear!a!sample!of!the!pure!culture!onto!a!piece!of!filter!paper.!
! Soak!the!colony!with!the!oxidase!reagent.!
! Wait!up!to!20!seconds.!Observe!for!color!change.!
o Results:!
! A!dark!blue!color!appearing!within!20!seconds!signals!a!positive!reaction.!!
! No!color!change!within!20!seconds!should!be!recorded!as!a!negative!
reaction.!
4.1$
!
o DO!NOT!use!colonies!from!MacConkey!agar!to!run!the!oxidase!test.!Growing!
colonies!will!pick!up!the!crystal!violet!dye!in!the!agar.!This!appears!pigmented!
against!the!white!filter!paper!and!may!be!interpreted!as!a!false!positive.!!
% Perform!3%!KOH!test!on!Bordetella!
o Purpose:!The!3%!KOH!test!is!a!rapid!biochemical!test!that!may!serve!as!a!
confirmation!of!your!Gram!stain!results.!It!may!NOT!be!used!to!replace!a!GramF
stain!!!
o How!it!works:!3%!KOH!works!by!lysing!the!thinner!cell!walls!of!GramFnegative!
bacteria,!releasing!chromosomal!DNA!into!the!emulsion.!This!leads!to!a!change!in!
viscosity.!GramFpositive!bacteria!will!not!lyse!and!will!therefore!not!result!in!a!
change!in!viscosity.!
o To!run!the!test:!!
1) Place!a!drop!of!3%!KOH!onto!a!clean!glass!slide.!
2) Pick!up!a!loopful!of!pure!culture!from!a!plate!and!emulsify!it!into!the!KOH.!
3) After!a!few!seconds,!carefully!lift!the!loop!a!few!centimeters!above!the!
emulsion!and!observe!for!a!clear!stringy!substance!connecting!the!loop!to!
the!emulsion.!!
o Results:!!
! GramFnegative:!the!emulsion!will!become!viscous!and!a!clear!stringy!
substance!will!be!observed!in!step!3.!
! GramFpositive:!there!will!be!no!change!in!viscosity!because!the!cell!walls!of!
GramFpositive!bacteria!are!too!thick!to!be!lysed!by!3%!KOH.!
% Pick!a!colony!from!the!SBA!plate!and!streak!it!onto!ReganFLowe!Charcoal!agar.!
o Purpose:!ReganFLowe!agar!is!a!selective!medium!designed!to!isolate!B.)pertussis.!
!
!
Day*3*
!
% No!additional!materials!needed.!
!
Procedure:!
% Observe!for!growth!on!ReganFLowe!agar.!If!no!colonies!are!found,!continue!to!incubate!the!
cultures.!!
% When!colonies!of!Bordetella!spp.!are!visible!to!the!unaided!eye,!they!should!appear!small,!
gray,!shiny,!convex,!smooth,!and!raised!with!a!pearlFlike!luster;!somewhat!like!a!mercury!
droplet!(Hardy!Diagnostics).!
!
!
*
*
*
*
*
4.2$
!
* Bordetella)pertussis)
*
Colony*
morphology*(on*
SBA*and*
MacConkey)*Note*
colony*color,*size,*
dimensions,* *************************** *
margin,*etc.* *
*
*
Gram*stain*(note*
GramIness,*cell*
morphology,*as*
well*as*cell*
arrangement)*
*
*
*
Oxidase*
*
*
3%*KOH*
*
*
ReganILowe*
Charcoal*agar*
(record*colony*
morphology,*#*
days*for*
incubation,*etc.)* *
*
*
4.3$
!
Experiment*5:*Identification*of*Corynebacterium.diphtheriae**
!
!
Day*1*
!
% 1!tube!of!pure!culture!of!C.#diphtheriae!
% 1!SBA!plate!
!
Procedure:!
% Direct!smear.!
% Streak!contents!of!the!tube!for!isolation!onto!SBA.!
!
!
Day*2*
!
% 1!Loeffler’s!slant!
% 1!Tinsdale!agar!
% S.#aureus!on!SBA!
!
Procedure:!
% Inoculate!Loeffler’s!slant!with!Corynebacterium.!
o Loeffler’s!slants!are!used!to!enhance!the!formation!of!metachromatic!granules!in!
corynebacteria.!It!is!used!in!conjunction!with!the!methylene!blue!stain!to!visualize!
these!granules.!!
% Split!a!Tinsdale!agar!plate!in!half.!Streak!half!with!the!Corynebacterium!isolate!and!the!
other!half!with#S.#aureus.!!
o Tinsdale!agar!is!a!biochemical!medium!used!for!the!screening!of!C.#diphtheriae.!The!
potassium!tellurite!in!the!medium!inhibits!gramInegative!bacteria!and!most!upper!
respiratory!tract!normal!flora.!Cystine!and!thiosulfate!serve!as!sources!of!sulfur!for!
reduction.!Once!reduced,!the!sulfur!will!react!with!the!potassium!tellurite!to!form!a!
metallic!precipitate.!This!metallic!precipitate!appears!as!a!brown!halo!surrounding!
the!colony.!!
o C.#diphtheriae!is!able!to!produce!a!brown!halo!around!each!colony!while!other!
bacteria!cannot.!!
!
!
Day*3*
!
% Methylene!blue!stain!
!
Procedure:!
5.1$
!
% Examine!Loeffler’s!slant!for!growth.!Perform!methylene!blue!stain.!Use!E.#coli!as!a!negative!
control.!!
o Make!a!smear!of!the!growth!as!you!would!for!a!GramIstain.!Once!it!has!airIdried,!
heatIfix!the!sample.!Flood!the!sample!with!methylene!blue!stain!for!30!seconds.!
Rinse,!blot!dry!and!examine!under!oil!immersion!for!metachromatic!granules.!!
% Examine!Tinsdale!agar!for!growth!and!the!presence!of!a!halo.!
!
!
* Corynebacterium.diptheriae.
*
Colony*morphology***
(on*SBA)*Note*colony*
color,*size,*dimensions,*
margin,*etc.*
*
*
*
Gram*stain*(note*GramG
ness,*cell*morphology,*as*
well*as*cell*arrangement)*
*
*
*
3%*KOH* *
*
*
Tinsdale*tellurite*agar*
(note*the*presence*or*
absence*of*a*halo)*
*
*
*
Loeffler’s*slant**
(note*the*presence*or*
absence*of*metachomatic*
granules)*
*
*
!
5.2$
!
Experiment*6:*Identification*of*Haemophilus,influenzae*
!
Haemophilus,influenzae!is!an!obligate!human!microorganism.!It!gets!its!name!“Haemophilus”!
(meaning!blood7loving)!from!the!fact!that!it!requires!specific!blood!factors!(X!and!V)!to!live!
(Leboffe!and!Pierce,!2011).!In!this!lab,!you!will!cultivate!Haemophilus!by!providing!these!factors!
in!various!ways.!!
! !
!
Day*1*
!
% Haemophilus,influenzae!on!chocolate!agar!
% S.,aureus!on!SBA!
% 1!SBA!plate!
% 1!Tryptose!agar!plate!
% X!factor!
% V!factor!
% X+V!factor!
!
Procedure:!
% Gram!stain!from!chocolate!agar.!
o Chocolate!agar!is!used!for!the!cultivation!of!fastidious!organisms!such!as!
Haemophilus!or!Neisseria.!It!is!compositionally!identical!to!SBA,!except!that!the!red!
blood!cells!have!been!lysed!with!heat.!This!lysing!of!the!red!blood!cells!releases!
hemin!(X!factor)!into!the!medium,!allowing!for!the!growth!of!H.,influenzae.!!
% Set!up!a!satellite!plate!
o Haemophilus!have!specific!nutritional!requirements.!They!cannot!thrive!without!a!
source!of!NAD!(V!factor)!and!hemin!(X!factor)!in!their!environment.!SBA!provides!
the!V!factor!but!the!X!factor!is!still!contained!within!the!red!blood!cells.!S.,aureus!
secretes!hemolysins!that!will!lyse!red!blood!cells,!releasing!X!factor!into!the!
medium.!In!the!presence!of!S.,aureus,!Haemophilus!can!grow!on!SBA.!This!
phenomenon!is!known!as!satellitism.!
o To!set!up!this!test,!create!a!lawn!of!Haemophilus!on!SBA!by!streaking!or!swabbing!
(Figure!6.1a).!Then!streak!S.,aureus!for!isolation!on!top!of!the!lawn.!
!!
! !!!! !!!!!$ !
!! !!!!Figure!6.1a! ! ! ! Expected!results!
6.1$
!
% Set!up!X!and!V!factor!test!
o The$nutritional$needs$of$Haemophilus$can$be$demonstrated$on$tryptose$agar.$
Tryptose$agar$lacks$both$the$X$and$V$factors.$These$factors$can$be$supplemented$
in$the$form$of$disks.$!
o To$set$up$this$test,$first,$inoculate$the$tryptose$agar$plate$by$evenly$spreading$
several$colonies$of$Haemophilus$onto$the$agar$surface.$Then$divide$the$plate$into$
quarters.$Aseptically$place$the$factor$disks$in$the$middle$of$each$quadrant$as$
shown$in$the$diagram$below$(Figure$6.2).$To$minimize$the$cross%diffusion$of$X$
and$V$factors$into$opposing$quadrants,$be$sure$to$place$the$“X$only”$and$“V$only”$
disks$on$opposite$sides$of$the$plate.$
$$ $
!! !!!!!!!Figure!6.2! ! ! !!!!!!!Expected!results!
% Incubate!plates!
o H.,influenzae!is!microaerophillic!and!grows!best!at!a!CO2!concentration!of!~5%.!To!
accomplish!such!environmental!conditions,!the!plates!will!be!incubated!in!a!candle!
jar.!!
!
!
Day*2*
!
% No!additional!materials!needed!
!
Procedure:!
% Examine!satellite!plate!
o Colonies!of!H.,influenzae!should!be!most!visible!in!the!hemolysis!zone!of!S.,aureus.!
% Examine!X!and!V!factor!test.!
o Growth!should!only!occur!in!the!presence!of!required!growth!factors.!
% Record!and!diagram!results!in!table!below.!!
!
!
!
!
!
!
!
!
6.2$
!
Experiment*6:*Identification*of*Haemophilus,influenzae*
! !
*
*
*
Colony*morphology*
!
*
*
*
Gram*stain*
!
*
!
Catalase*
*
*
!
3%*KOH*
*
* !
!
Satellite*plate*
* !
* !
* !
!
X*and*V*factors*
* !
* !
6.3$
!
Experiment*7:*Enterobacteriaceae*
*
Day*1*
!
4 1!tube!of!mixed!culture!enterics!(Salmonella!+!E.*coli!+!Pseudomonas)!
4 1!XLT44!plate!
4 1!HE!plate!
! !
Procedure:!
4 Direct!smear!of!mixed!culture!enterics.!
4 Streak!contents!of!the!tube!for!isolation!on!XLT44!and!HE!agar.!
o Several!types!of!media!have!been!developed!over!the!years!specifically!for!the!
isolation!and!identification!of!Salmonella*sp.!Media!such!as!XLD!(Xylose!Lysine!
Deoxycholate),!XLT44!(Xylose!Lysine!Tergitol!4)!and!HE!(Hektoen!Enteric)!agar!
will!screen!for!Salmonella!by!taking!advantage!of!its!ability!to!reduce!thiosulfate!
to!hydrogen!sulfide!gas!(H2S).!The!H2S!gas!produced!will!react!with!iron!
supplied!by!ferric!ammonium!citrate!to!produce!a!black!precipitate.!The!
precipitate!is!limited!to!the!colonies!and!does!not!spread!out!into!the!
surrounding!agar.!As!a!result,!Salmonella*colonies!will!appear!jet!black.!On!SBA,!
Salmonella*look!like!all!other!enterics!but!on!XLT44!and!HE,!they!grow!as!black!
(H2S!producing)!colonies.!!
o XLT44!is!used!for!the!isolation!of!non4typhi*Salmonella!species.!Tergitol!4!
inhibits!all!Gram4positive!bacteria!and!molds.!It!also!inhibits!the!growth!of!
numerous!Gram4negative!bacteria,!including!Proteus,*Providencia!and!
Pseudomonas.!The!medium!contains!lactose,!sucrose!and!xylose.!Enterics!that!
produce!acid!from!fermentation!will!produce!yellow!colonies.!Organisms!that!
do!not!ferment!the!sugars!will!produce!pink!colonies.!Because!Salmonella!
species!reduce!thiosulfate!but!do!not!ferment!the!sugars,!they!will!appear!as!
black!colonies!with!pink!edges.!The!only!drawback!of!using!XLT44!is!that!Group!
D!Salmonella!are!not!easily!identified!on!this!medium.!
o HE!agar!is!a!selective!medium,!similar!to!XLT44!agar.!Bile!salts!are!included!to!
inhibit!most!Gram4positive!cocci.!The!medium!contains!lactose,!sucrose!and!
salicin.!Enterics!that!produce!acid!from!fermentation!will!produce!yellow!to!
salmon4pink!colonies.!Organisms!that!do!not!ferment!any!of!the!sugars!will!
produce!blue4green!colonies.!Group!D!Salmonella,!a!group!of!strains*that!are!not!
as!easily!selected!for!on!the!XLT44!medium,!is!more!easily!detected!with!HE!
agar.!The!method!of!screening!for!suspected!Salmonella*strains!is!the!same—
black!colonies!indicate!a!possible!Salmonella*identification.!Because!Salmonella!
cannot!ferment!any!of!the!sugars,!the!black!colony!will!have!a!blue4green!edge.!
o * Enrichment!broths!are!used!to!detect!bacteria!that!may!be!present!in!low!
numbers!in!the!sample!relative!to!other!bacteria.!A!selective!enrichment!
medium!creates!an!environment!that!is!favorable!to!the!growth!of!your!target!
organism!while!suppressing!competition!from!other!bacteria.!Selenite!broth!is!
used!to!enrich!for!Salmonella*because!selenite!has!been!discovered!to!
7.1!
!
temporarily!inhibit!the!growth!of!other!bacteria!for!up!to!18,!but!no!more!than!
24,!hours.!However,!at!least!100!Salmonella/gram!of!feces!are!needed!for!a!
successful!enrichment.!Selenite!inhibits!the!growth!of!coliforms!for!up!to!12!
hours,!allowing!Salmonella*to!replicate!without!competition.!Ideally,!subcultures!
will!be!made!in!the!first!12418!hours!for!maximum!Salmonella*recovery.!The!
enrichment!is!void!after!24!hours.!
!
!
Day*2*
*
4 2!SBA!plates!
4 2!MacConkey!agar!plates!
4 2!tubes!of!pure!culture!enteric!(E.*coli,*Klebsiella,*Proteus,*Serratia!or!Yersinia)!or!
Pseudomonas*
*
Procedure:!
4 Continue!from!day!1:!
o Observe!XLT44!and!HE!plates!for!growth.!Record!colony!morphologies.!
o Salmonella!colonies!should!appear!jet!black.!(The!yellow!colonies!are!E.*coli.!
They!are!yellow!due!to!the!fermentation!of!sugars!in!the!media.!They!are!not!
black!because!they!do!not!reduce!thiosulfate.)!
o Pick!one!isolated!Salmonella!colony!from!either!the!HE!or!the!XLT44!plate!and!
streak!it!for!isolation!on!both!SBA!and!MacConkey.!
4 Direct!smear!of!pure!culture!enteric!or!Pseudomonas.!
4 Streak!contents!of!the!tube!for!isolation!on!both!SBA!and!MacConkey.!
o MacConkey!agar!is!used!to!isolate!and!differentiate!members!of!
Enterobacteriaceae!based!on!their!ability!to!ferment!lactose!(Leboffe!and!Pierce,!
2011).!Bile!salts!and!crystal!violet!inhibit!the!growth!of!Gram4positive!bacteria!
and!some!Gram4negative!bacteria!with!the!exceptions!of!Pseudomonas!and!
Bordetella*spp.!The!pH!indictor,!neutral!red,!is!red!below!the!pH!of!6.8.!
Organisms!capable!of!fermenting!lactose!will!appear!a!shade!of!red!whereas!
non4lactose!fermenters!will!remain!their!normal!color!or!take!on!the!color!of!the!
medium.!!
!
!
Day*3*
!
Materials:!(per!enteric)!
4 Oxidase!
4 TSI!slant!
4 SIM!tube!
4 Urea!slant!!
4 Citrate!slant!
!
Procedure:!
7.2!
!
4 Examine!plates!for!purity.!Confirm!colony!morphologies.!Record!results.!
4 Perform!Gram!stain.!
4 Perform!oxidase!test.!!
o Be!sure!to!use!a!colony!from!SBA.!The!pigments!of!colonies!on!MacConkey!agar!
can!be!mistaken!as!a!false!positive.!!
4 Inoculate!TSI,!SIM,!Urea,!Citrate!and!O/F!tubes!for!each!enteric!(or!Pseudomonas).!!
o TSI!(Triple!Sugar!Iron)!slant:!TSI!is!a!rich!medium!designed!to!differentiate!
enterics!based!on!glucose!fermentation,!lactose!fermentation,!sucrose!
fermentation,!thiosulfate!reduction!and!gas!production.!The!medium!is!
prepared!as!a!slant,!providing!both!an!aerobic!and!an!anaerobic!environment.!
The!test!is!to!be!inoculated!by!stabbing!the!agar!butt!followed!by!streaking!of!
the!slant.!!!
o SIM!(Sulfur,!Indole,!Motility):!This!medium!is!a!semi4solid!medium!containing!
casein!(a!source!of!amino!acids),!iron!and!sodium!thiosulfate.!It!is!designed!to!
test!for!thiosulfate!reduction,!indole!production,!and!motility.!The!test!is!to!be!
inoculated!by!inserting!a!stab!straight!down!into!the!agar.!The!low!percentage!
of!agar!creates!a!semi4solid!medium!that!allows!for!the!movement!of!motile!
bacteria.!Motility!is!observed!by!a!fuzzy!growth!beyond!the!stab.!Some!strains!
will!be!so!motile!that!the!entire!tube!will!appear!homogenous.!It!may!help!to!
compare!the!results!to!an!uninoculated!tube.!(One!thing!to!keep!in!mind!is!that!
although!Pseudomonas!is!a!motile!bacterium,!it!will!appear!non4motile!with!the!
SIM!test!due!to!its!inability!to!grow!under!anaerobic!conditions.)!If!the!organism!
can!reduce!thiosulfate,!the!H2S!produced!will!react!with!the!iron!in!the!medium!
to!form!a!black!precipitate.!The!black!precipitate!can!deposit!in!2!ways:!on!the!
track!of!inoculation!(thin!black!line!=!non4motile)!or!throughout!the!agar!plug!
(black!throughout!=!motile).!The!indole!test!is!performed!by!adding!Kovac’s!
reagent!to!the!tube!and!watching!for!the!formation!of!a!pink!color.!Some!
bacteria!possess!the!ability!to!produce!the!enzyme!tryptophanase,!which!
hydrolyzes!tryptophan!(an!amino!acid)!to!indole;!indole!reacts!with!Kovac’s!
reagent!to!form!a!dye!that!is!red!in!color.!!
o Urea!slant:!This!medium!is!used!to!differentiate!between!organisms!based!on!
their!ability!to!hydrolyze!urea.!The!pH!indicator,!phenol!red,!is!yellow!or!orange!
below!pH!8.4!and!red!or!pink!above!pH!8.4.!Urea!hydrolysis!to!ammonia!will!
make!the!medium!alkaline,!turning!the!pH!indicator!pink.!The!test!is!to!be!
inoculated!by!streaking!the!slant.!
o Citrate!slant:!This!medium!tests!for!the!ability!of!an!organism!to!utilize!citrate!as!
the!sole!carbon!source.!Citrate!is!added!into!the!medium!as!sodium!citrate.!In!
solution,!sodium!citrate!dissociates!into!sodium!and!citrate!ions.!(If!you!
remember!from!chemistry,!water!also!will!also!self4ionize!into!H3O+!and!OH4!
ions.)!If!a!cell!is!able!to!transport!citrate!into!the!cell,!it!will!transport!it!along!
with!H+!ions!to!balance!out!the!negative!charge.!As!a!result,!the!uptake!of!citrate!
will!result!in!an!increase!in!the!pH!of!the!medium.!The!pH!indicator,!
bromothymol!blue,!is!green!at!neutral!pH!(~6.9)!and!blue!at!a!more!alkaline!pH!
(~7.6).!A!blue!slant!indicates!a!positive!test.!The!test!is!to!be!inoculated!by!
streaking!the!slant.!
7.3!
!
o O/F!tubes:!O/F!tubes!are!used!to!differentiate!bacteria!based!on!their!ability!to!
oxidize!or!ferment!specific!sugars.!The!sugar!sources!can!vary.!In!this!class,!we!
will!be!using!glucose.!Prior!to!inoculation,!the!tubes!should!be!boiled!and!cooled!
to!remove!the!free!oxygen!from!the!medium.!Once!cooled,!the!test!is!to!be!
inoculated!by!inserting!a!stab!straight!down!into!the!agar.!Do!this!for!2!tubes.!
One!tube!will!be!incubated!as!is.!The!other!tube!will!be!topped!with!
approximately!0.5!cm!(height!in!tube)!of!sterile!mineral!oil!to!promote!
anaerobic!growth!and!fermentation.!The!pH!indicator,!bromothymol!blue,!is!
green!at!neutral!pH!(~7)!and!yellow!at!an!acidic!pH!(~!6.0).!The!fermentation!of!
glucose!will!result!in!acidic!byproducts!that!will!turn!the!pH!indicator!yellow.!
!
!
Day*4*
!
4 Kovac’s!reagent!(indole)!
4 Antigen!agglutination!test!for!Salmonella!(if!applicable)!
!
Procedure:!
4 Observe!biochemical!tests.!
o When!reading!TSI’s,!the!first!thing!to!do!is!to!check!the!acidity!of!the!slant!and!
the!acidity!of!the!butt.!The!pH!of!the!slant!represents!aerobic!metabolism!while!
the!pH!of!the!butt!represents!anaerobic!metabolism.!The!acidity!of!these!two!
portions!can!be!readily!distinguished!by!the!color!changes!of!the!pH!indicator,!
phenol!red.!Red!represents!alkaline!byproducts!while!yellow!and/or!black!
represent!acidic!byproducts.!Next,!check!for!H2S!production.!If!the!butt!portion!
of!the!tube!is!visibly!black,!then!the!bacterium!is!H2S!positive!(and!has!an!acidic!
butt).!Finally,!examine!the!tube!for!gas!production.!Sometimes!gas!production!is!
not!obvious!and!careful!inspection!is!needed.!Other!times,!there!is!so!much!gas!
production!that!the!agar!plug!is!pushed!away!from!the!bottom!of!the!tube.!!!
4 Perform!antigen!agglutination!test!for!Salmonella.!
o The!antigen!agglutination!test!is!a!rapid!biochemical!test!that!can!confirm!or!
rule!out!the!identity!of!your!isolate.!If!the!O!antigen!is!present,!antibodies!in!the!
test!will!bind!to!the!antigens,!causing!visible!clumps!to!form.!First,!grab!a!clean!
glass!slide!and!draw!a!nickel4sized!circle!on!the!slide!with!a!wax!crayon.!This!
will!confine!the!reaction.!Next,!add!a!drop!of!reagent!inside!the!circle.!Pick!up!a!
loopful!of!pure!culture!and!homogenize!it!with!the!reagent.!Gently!rock!the!slide!
back!and!forth!and!observe!for!any!changes.!If!your!isolate!is!Salmonella,!the!
mixture!should!change!from!a!milky4white!solution,!to!a!clear!solution!topped!
with!small!white!clumps.!A!negative!reaction!will!yield!no!change!and!will!
remain!milky4white.
7.4!
!
Experiment*7:*Enterobacteriaceae*
! Pseudomonas! E.!coli! Klebsiella! Proteus! Salmonella! Serratia! Shigella! Yersinia!
* * * * * * * *
Gram*stain*
*
* * * * * * * *
Colony* *
morphology* *
(on*SBA*and* *
MacConkey)* *
For$ *
Salmonella,$
*
include$colony$
morphology$on$ *
XLT54$and$HE.$ *
*
* * * * * * * *
Oxidase*
*
* * * * * * * *
Indole*
*
* * * * * * * *
SIM*
*
* * * * * * * *
*
TSI* *
*
*
* * * * * * * *
Urea*
*
* * * * * * * *
Citrate*
*
$
EnteroPluri(Test
Sector Group Biochemical0Reaction Positive Negative Results Score01 Total01
Glucose0fermentation03 Yellow04 Red04 2
Glucose/Gas 1
Gas0production03 Wax0lifted Wax0not0lifted 1
Lysine Lysine0decarboxylation Violet Yellow 4
Ornithine 2 Ornithine0decarboxylation Violet Yellow 2
H2S0production03 BlackJbrown Beige 1
H2S/Indole
Indole0test03 PinkJred Colorless 4
Adonitol 3 Adonitol0fermentation Yellow Red 2
Lactose Lactose0fermentation Yellow Red 1
Arabinose Arabinose0fermentation Yellow Red 4
Sorbitol Sorbitol0fermentation Yellow Red 2
4
VP02 Acetoin0production Red Colorless
Dulcitol0fermentation Yellow Green 1
Ducitol/PA
Phenylalanine0deamination Dark0brown Green 4
Urea 5 Urea0hydrolysis03 Purple04 Beige04 2
Citrate Citrate0utilization03 Blue Green 1
Reading(the(test:
1
0The0150biochemical0tests0are0divided0into050groups.0Each0test0has0a0positivity0value0of04,020or01.0The0negativity0value0of0all0tests0is0
zero.0If0the0test0result0is0positive,0circle0the0corresponding0number0in0the0"score"0column.0If0the0test0result0is0negative,0draw0an0"X"0
through0the0corresponding0number0in0the0"score"0column.0Perform0the0indole0test0(by0adding0Kovac's0reagent0to0the0H2S/Indole0
sector)0after0all0other0tests0have0been0observed.0Total0the0values0of0all0of0the0positive0tests0in0each0group.0The0end0result0is0a05J
digit0code0that0allows0for0the0identification0of0the0organism.0
2
0The0EnteroPluri0test0can0be0scored0in0the0absence0of0VP0results.0However,0the0respective0database/codebook0must0be0used.
A(few(notes:(
3
0Some0of0the0biochemical0tests0listed0should0be0familiar.0The0ones0labelled0have0been0independently0tested0in0Lab050through0tests0
such0as0O/F0tubes,0TSI0slants,0SIM0tubes,0urea0slants0and0citrate0slants.
4
0The0results0here0appear0different0from0those0found0in0the0independently0inoculated0tests.0This0is0simply0a0difference0in0the0pH0
indicator0used.0The0test0itself0functions0the0same0way.
Experiment*10:*Got*food*poisoning?*Could*B.#cereus.*
!
!
Day*1*
!
% 2!tubes!of!pure!culture!(Bacillus(cereus!and!Bacillus(subtilis)!
% 1!SBA!
% 1!Mannitol!Yolk!Polymyxin!B!(MYP)!plate!
!
Procedure:!
% Direct!smear.!
% Streak!the!contents!of!the!tube!for!isolation!on!SBA.!!
% Streak!the!contents!of!the!tube!for!isolation!on!MYP.!
o Mannitol!Yolk!Polymyxin!B!is!a!selective!and!differential!medium!used!for!the!
isolation!of!B.(cereus!from!food.!Polymyxin!B!is!an!antibiotic!that!inhibits!the!growth!
of!most!other!bacteria.!Mannitol!is!available!as!a!fermentable!carbon!source;!those!
who!can!ferment!mannitol!will!lower!the!pH!of!the!medium,!resulting!in!a!yellow!
color.!B.(subtilis!can!ferment!mannitol!while!B.(cereus!cannot.!Egg!yolk!emulsion!in!
the!medium!provides!lecithin.!Lecithinase!positive!colonines!will!hydrolyze!the!
lecithin,!forming!a!zone!of!white!precipitate!around!the!colony.!
!
*
Day*2*
!
% 2!SIM!tubes!
!
Procedure:!
% Examine!and!record!colony!morphologies.!
% Perform!catalase!test.!
% Inoculate!1!SIM!for!each!unique!colony!type!observed.!
!
!
Day*3*
!
% No!additional!materials!needed.!
!
Procedure:!
% Record!SIM!results.!
!
!
!
10.1$
!
!
Experiment*10:*Got*food*poisoning?*Could*B.#cereus.*
! Bacillus#cereus# Bacillus#subtilis#
!
Colony*morphology*on*SBA*
Note*colony*color,*size,*
dimensions,*margin,*etc.*
! !
!
!
Gram*stain*
dimensions,*margin,*location*
of*spore*etc.*
! !
!
!
Spore*location* !
!
!
Catalase* !
!
!
Motility*(SIM)* !
!
!
Colony*morphology*on*MYP*
! !
!
!
Mannitol*fermentation* !
!
!
Lecithinase* !
!
!
10.2$
!
Experiment*11:*Antibiotic*Sensitivity*
!
*
Introduction*
*
It!was!once!believed!that!infectious!diseases!would!be!eliminated!through!the!use!of!
antimicrobials.!Unfortunately,!this!optimism!was!halted!by!the!development!of!antibiotic!
resistance.!The!Kirby!Bauer!Disk!Diffusion!Assay!allows!microbiologists!to!test!the!efficacy!of!
multiple!antibiotics!on!one!culture.!This!data!assists!physicians!in!the!selection!of!treatment!
options.!!
!
The!Kirby!Bauer!disk!diffusion!assay!has!been!standardized!to!ensure!uniformity!of!technique!
and!reproducibility!of!results.!This!includes,!but!is!not!limited!to!the!following:!
• Each!disk!has!been!impregnated!with!a!known!concentration!of!an!antimicrobial!
compound.!When!placed!onto!the!plate,!the!antimicrobial!compound!in!the!disk!will!
diffuse!out!into!the!surrounding!agar.!The!area!closest!to!the!disk!will!contain!a!higher!
concentration!of!the!antimicrobial.!Each!antimicrobial!has!a!different!minimum!
inhibitory!concentration!(MIC)!resulting!in!varying!zones!of!inhibition.!
• Each!plate!has!been!poured!to!a!specific!depth!of!4!mm.!Because!the!antimicrobial!
diffuses!in!a!3Ldimensional!fashion,!shallow!agar!layers!will!produce!larger!zones!of!
inhibition!than!deep!agar!layers.!!
• The!turbidity!of!each!sample!should!resemble!the!turbidity!of!a!0.5!MacFarland!
standard.!The!MacFarland!standard!allows!for!the!visual!comparison!of!bacterial!
density.!This!controls!the!size!of!the!inoculum.!!
*
*
Day*1*
!
$ 2!MuellerLHinton!broths!
$ S.#aureus!(saved!from!nasal!culture!in!Experiment!2)!
$ E.#coli#
!
Procedure:!
$ Using!a!sterile!loop,!inoculate!a!tube!of!MuellerLHinton!broth!with!a!sample!of!S.#aureus.!!
$ Repeat!in!the!second!tube!with!E.#coli.!!
!
!
Day*2*
!
$ 0.5!MacFarland!Standard!
$ 2!sterile,!disposable,!plastic!pipets!
! 11.1#
$ 2!tubes!of!sterile!water!
$ 2!sterile,!cottonLtipped!applicators!
$ 2!MuellerLHinton!agar!plates!
$ Antibiotic!disks!(2!each!of!ampicillin,!chloramphenicol,!kanamycin,!penicillin,!and!
trimethoprim)!
!
Procedure:!
$ Set!up!Kirby!Bauer!Disk!Diffusion!Assay!
o Gently!shake!the!overnight!cultures!to!resuspend!the!bacteria!in!broth.!This!is!your!
stock!culture.!
o Using!the!sterile!disposable!pipets,!dilute!the!stock!cultures!in!sterile!water,!1!drop!
at!a!time,!until!the!turbidity!reaches!that!of!the!0.5!MacFarland!standard.!
! It!is!important!to!resuspend!the!particles!in!the!MacFarland!standard!before!
using!it!for!comparison.!
o Saturate!a!sterile!cotton!swab!with!the!newlyLmade!suspension.!Gently!wring!out!
the!excess!liquid!by!rolling!the!swab!against!the!side!of!the!tube.!!
o Paint!a!lawn!on!the!plate!by!swabbing!back!and!forth!from!top!to!bottom.!Rotate!
the!plate!90º!and!repeat.!Rotate!the!plate!90º!and!repeat!a!second!time.!This!
ensures!an!even!lawn!of!growth.!DO!NOT!resaturate!the!swab!in!between!streaks!!
o Allow!the!liquid!on!the!plate!to!dry.!
o Using!sterile!forceps,!aseptically!place!5!evenlyLspaced!antibiotic!disks!on!the!agar!
surface!and!gently!tap!the!disks!in!place!with!forceps.!!
! Do!not!put!the!disks!too!close!to!the!edge.!The!antibiotic!will!not!diffuse!
properly.!It!will!also!make!it!more!difficult!to!measure!zones!of!clearing.!
! Gently!tap!the!antibiotic!disks!into!place!to!ensure!complete!contact!of!the!
disk!with!the!agar!surface.!!
!
!
Day*3*
!
$ Ruler#
!
Procedure:!
$ Measure!the!zones!of!clearing!(in!mm).!Record!and!compare!the!results.!!
!
!
! 11.2#
Kirby&Bauer&Disk&Diffusion&Assay
Disk3 Disk3 Zone3diameter3(mm) Results
Antimicrobial3agent Effect3on3cells
Identifier content Resistant Intermediate Susceptible S.#aureus E.#coli
Ampicillin31 AM610 103µg Bactericidal
##.#Enterobacteriaceae ≤313 14616 ≥317
##.#Staphylococcus ≤328 – ≥329
Chloramphenicol323 C630 303µg Bacteriostatic
##.#Staphylococcus ≤312 13617 ≥318
Kanamycin333 K630 303µg Bactericidal
Penicillin31 P610 103units Bactericidal
##.#Enterobacteriaceae – – –
##.#Staphylococcus ≤328 – ≥329
Trimethoprim343 TMP65 53µg Bacteriostatic
Mode3of3action:
1
3Ampicillin3is3a3type3of3penicillin,3differing3from3the3latter3by3the3presence3of3an3amino3group.3This3amino3group3allows3the3
antibiotic3to3penetrate3Gram6negative3outer3membranes3whereas3penicillin3is3only3effective3against3Gram6positive3bacteria.3
Ampicillin3and3penicillin3are3both3in3the3beta6lactam3group3of3antibiotics.3Beta6lactams3competitively3inhibit3transpeptidase,3which3
is3needed3to3form3the3crosslinks3in3peptidoglycan3layers.3This3eventually3leads3to3the3activation3of3hydrolases3which3result3in3cell3
lysis,3hence3the3bacteriocidal3effects3of3beta6lactams.
2
3Chloramphenicol3works3by3binding3the350S3ribosomal3unit3of3bacteria3and3inhibiting3peptide3bond3formation.3This3effect3is3
bacteriostatic3but3may3be3bacteriocidal3in3higher3concentrations.3
3
3Kanamycin3is3in3the3aminoglycoside3group3of3antibiotics.3Aminoglycosides3work3by3binding3to3the330S3ribosomal3unit3to3inhibit3
protein3production.3However,3kanamycin3works3by3causing3frameshift3mutations3so3that3instead3of3halting3protein3production,3
misfolded3or3incorrect3proteins3are3produced,3eventually3leading3to3cell3death.3
4
3Trimethoprim3is3part3of3the3chemotheraputic3class3of3antibiotics.3Trimethoprims3work3by3inhibiting3folic3acid3production;3more3
specifically,3dihydrofolate3reductase.3This3affects3the3production3of3the3nucleotide3thymidine3and3other3amino3acids3which3are3
necessary3for3DNA3replication.3Without3it,3cells3are3unable3to3replicate3and3so3are3unable3to3further3affect3the3host.3
Experiment*12:*Fungus*is*among*us*
!
!
Day*1*
!
% 1!tube!of!mixed!culture!(yeast!mix!+!S.#aureus)!
!
Procedure:!
% Direct!smear!
o Yeast!cells!can!be!mistaken!as!Gram@positive!cocci.!However,!their!large!size!will!
give!them!away.!S.#aureus!has!been!included!as!a!point!of!reference!for!the!yeast.!
Compare!their!sizes.!!
!
!
Day*2*
!
% Cryptococcus#neoformans!on!Potato!Flake!agar!
% Cryptococcus#neoformans!on!Cornmeal!agar!with!Tween!80!
% Candida#albicans!on!Potato!Flake!agar!
% Candida#albicans!on!Cornmeal!agar!with!Tween!80!
!
Procedure:!
% Examine!and!record!colony!morphologies.!!
o Potato!Flake!agar!to!yeast!is!like!blood!agar!to!bacteria!–!it!is!an!all@purpose!medium!
for!the!culture!of!yeast.!Potato!extracts!are!the!primary!nutrient!source.!
o Cornmeal!agar!with!Tween!80!is!a!medium!used!to!demonstrate!pseudohyphae!
formation!in!yeasts.!Tween!80,!a!mild!detergent,!is!added!to!the!agar!and!works!by!
stressing!the!cells!out!enough!to!induce!pseudohyphae!formation.!The!
pseudohyphae!are!visible!through!the!transparent!agar!and!are!used!to!distinguish!
Candida,!which!can!form!pseudohyphae!from!Cryptococcus,!which!cannot.!!
! Remove!the!lid!from!the!Cornmeal!agar!plate!and!place!the!plate!on!the!
microscope!platform.!Carefully!bring!the!colonies!into!focus!under!10X!to!
see!the!pseudohyphae!in!greater!detail.!
% The!India!Ink!stain!is!used!to!visualize!capsules.!The!ink!particles!are!too!large!to!penetrate!
the!polysaccharide!capsule,!resulting!in!white!unstained!circles!surrounded!by!a!dark!
background.!Cryptococcus!produces!a!capsule!in!vivo!but!this!capsule!is!often!lost!in!vitro.!
Use!the!cornmeal!agar!plates!to!perform!the!India!Ink!stain.!!
!
!
!
!
!
12.1$
!
Experiment*12:*Fungus*is*Among*us*
! Candida!albicans! Cryptococcus!neoformans!
!
Colony*morphology*on*
Potato*Flake*agar**
#*days*of*growth:*___*
! !
!
!
Colony*morphology*on*
Cornmeal*agar*with*1%*
tween*80*
#*days*of*growth:*___* ! !
!
!
Gram*stain*
(note*GramKness,*cell*
morphology,*cell*size,*etc.)*
! !
!
!
India*ink*stain*
! !
!
!
Pseudohyphae*formation*
! !
!
!
!
!
12.2$
!
Microbes
Microbes Page,#
Aerobic/anaerobic,,endospore4forming,bacilli
!"!Bacillus!cereus 2
!"!Clostridium!perfringens!Type&A 3
Aerobic/anaerobic,bacteria, 4
Aerobes,
!"!Bordetella! 5
!"!Legionella!pneumophila 6
!"!Mycoplasma!pneumoniae 7
!"!Neisseria 8
!"!Pseudomonas!aeruginosa! 9
Facultative,anaerobes,
!"!Corynebacterium &10111
!"!Enterobacteriaceae 12
!!!!!! E.!coli 13
!!!!!! Klebsiella!pneumoniae 14
!!!!!! Salmonella 15116
!!!!!! Vibrio!parahaemolyticus 17
!!!!!! Yersinia! 18
&1&Staphylococci 19120
&1&Streptococci 21122
Microaerophiles
!"!Campylobacter 23
!"!Haemophilus!influenzae 24
!"!Helicobacter 25
Acid4fast,organisms
!"!Mycobacterium 26127
!"!Norcardia 28
Non4spore,forming,Gram4negative,pleomorphics
!"!Bacteroides!fragilis 29
!"!Prevotella!intermedia 29
!"!Fusobacterium!nucleatum 29
Non4spore,forming,Gram4positive,coccobacilli,,pleomorphic
!"!Propionibacterium!acnes 30
!"!Actinomyces 30
Pathogenic,Fungi
&1&Dermatophytes 31
Pathogenic,Yeasts
!"!Candida!albicans 32
!"!Cryptococcus!neoformans 33
Spirochetes 35136
!"!Borrelia 35136
!"!Leptospira 36
!"!Treponema 36
AEROBIC/ANAEROBIC, ENDOSPORE-FORMING BACILLI
!
!
! Bacillus cereus
Characteristics
Gram-positive, aerobic, large rods of 1-1.3µm x 3-10µm with square or concave ends. Ovoid,
subterminal spores that appear unstained in a gram stain. Will form spores under aerobic
conditions (unlike Clostridium). Colony morphology: variable in size, raised, irregular with a
grayish to greenish frosted-glass appearance and undulate margins. Hemolytic on blood agar.
!
Ecology/Transmission
- Ubiquitous, found in soil, water, airborne dust. Some species are part of the normal intestinal,
oral flora.
- Toxin-mediated food poisoning
o Emetic toxin – vomiting
!! Heat-stable
!! 1-6 hours after ingestion
!! Room temp. stored rice, vegetables, cereals, powered milk
o Enterotoxin - associated watery diarrhea
!! Heat-labile
!! 10-12 hours after ingestion
!! Poultry, cooked meats, mashed potatoes, soups and desserts
- Demonstrated by finding >105 bacteria/gram of food
!
Diagnostic tests
a. Colony Morphology
b. Catalase Positive
c. Lecithinase
d. Hemolysis
e. Motility
!
To eliminate the possibility of the organisms being contaminants or opportunistic pathogens,
several laboratory observations can be made:
a. Presences of Bacillis colonies on the streak lines, not outside the lines on the culture plate
b. Isolation of the same species from 2 or more blood cultures from different venipuncture
sites
c. Repeated isolation from normally sterile sites.
d. Presence of morphological forms of the organism in addition to acute inflammatory cells
in a direct smear that was also culture positive.
e. Clinical evidence if infection in a compromised patient that is not explained by infection
with another bacteria.
! Microbes!–!page!2!
!
! Clostridium perfringens Type A
Characteristics
- Moderate oxygen tolerance, microaerophilic
- Non-motile gram positive box car shaped rod
- Central spores formed (generally not seen). Can be induced by growing on a chopped meat
media grown anaerobically for 5-7 days at 30oC
- Flat rhizoid centrally raised colonies
- Double zone of hemolysis on blood agar
- Normal intestinal flora -many isolated in a clinical setting may be contaminates,
therefore pathogenicity is dependent on the location of sample
- Produces myonecrosis (gas gangrene)
- 3rd most common for food poisoning 7-30 hour onset
- Antibiotic-associated diarrhea
- Necrotizing enterocolitis
!
Diagnostic Tests
The clinical specimen contains large, gram-positive rods with blunted ends in a necrotic
background; few leukocytes; suspected gas gangrene.
1. Liquefies gelatin but coagulated egg or serum is not digested.
2. Indole, catalase, lipase, H2S2, negative
3. Stormy milk reaction (proteolysis of milk)
4. Acid and gas in glucose, maltose, lactose, sucrose
5. Demonstration of alpha toxin hot-cold lysis (reverse CAMP test)
6. Lecithinase
7. Stain and motility
8. Lipase
9. Cytotoxin assay-Nagler Assay
!
Others of importance: C. botulinum (food poisoning), C. tetani, C. difficile (antibiotic associated
colitis, hospital acquired colitis, pseudomembranous colitis)
AEROBIC/ANAEROBIC BACTERIA
!
Characteristics
Able to grow in the absence of free molecular oxygen. Can obtain energy through fermentative
pathways where organic acids, alcohols or other products serve as the final electron acceptor.
!
Classification
1. Obligate anaerobes- 2 subdivisions
a. Strict
- Cannot grow on agar exposed to O2 above 0.5%
- Killed if exposed to atmosphere >10 minutes
- Rare in human infections
- Part of the normal flora
b. moderate
- Can grow when exposed to 2-8% O2
- Rare in human infections
- Part of the normal flora
2. Aerotolerant anaerobes
- Limited growth in room air or in 5-10% CO2
3. Facultative anaerobes
- Can exploit either condition for growth by using oxygen as a terminal acceptor or by
fermentation
4. Microaerophiles
- Require oxygen as the terminal electron acceptor but will not grow on the surface of
solid media in a standard incubator and will grow minimally under anaerobic
conditions.
- Do not grow on the surface of agar in an aerobic incubator (21% 02) and barely grows
anaerobically
!
Habitats
- Soil, marshes, oceans, sewage, foods and animals
- Humans = oral cavity, GI tract, genitourinary orifices and skin
- out number coliforms 1000:1
!
General Characteristics for Identification
1. Relation to O2
2. Colony morphology
3. Gram stain, microscopic features
4. Motility
5. Growth on liquid media
6. GLC (gas liquid chromatography). Refer to page 81
7. Antibiotic susceptibility
8. Serological tests
Microbes!–!page!4!
!
AEROBES
!
Bordetella
!
Bordetella pertussis
Characteristics
Gram-negative coccobacilli, non-motile, obligate aerobes, extracellular, polysaccharide capsule;
facultative anaerobe
!
Ecology/transmission
Humans are the only reservoir (B. pertussis)
- Highly contagious via respiratory droplets
- Attack rates of 90% among non-immune
- Atypical/mild unrecognized pertussis helps to disseminate the disease because the
communicability is greatest when the bacilli are present in the greatest numbers which is
during the catarrhal and early paroxysmal stages
!
Clinical presentation
Whooping cough aka Acute Tracheobronchitis
- Complications are: aspiration pneumonia, physical injuries/ CNS abnormalities/seizure as a
consequence of severe coughing
- Most deaths occur in unvaccinated children under the age of 5 years
- Immunity develops with recovery
!
Diagnostic tests
1. Gram Stain: small, pale-staining gram-negative coccobacilli which become more
pleomorphic on subculture, (may appear almost as cocci in single pairs; or pairs end to
end that resemble chains).
2. Colony Morphology/ Growth: specimen is from a nasal-pharyngeal swab
a. Slow-growing (2-4 days)
b. Narrow zone of hazy hemolysis on sheep blood agar
c. “Mercury droplet” colonies on selective media, Bordet-Gengou potato infusion agar
(starch neutralizes toxic materials)
d. Regan-Lowe medium (charcoal agar with horse blood) helps neutralize toxic
materials such as unsaturated fatty acids, sulfides and peroxides in agar or specimen,
available as transport and enrichment medium; white mother-of-pearl opalescence
colonies; incubate in CO2 enriched environment, 35oC; necessary to hold plates at
least 7 days
3. Oxidase Positive
4. Direct Fluorescent Antibody (DFA)
5. Serological Tests: Western Blot and Enzyme-linked Immunoassay
6. PCR
!
Legionella pneumophila
!
Characteristics
Gram-negative thin rods, aerobic, intracellular; motile, monotrichous flagella; non-encapsulated
!
NOT person to person transmission
- Contaminated water i.e. air conditioners, whirlpools, humidifiers; inhalation of aerosols,
aspiration from contaminated water; etc.
!
Legionnaires Disease
- Atypical pneumonia = non-productive cough, fever-> fatal in elderly and the Immune
compromised; nosocomial /community acquisition.
!
Diagnostic tests
1. Catalase positive
2. DFA
3. Urinary antigen test
4. Gram stain shows gram-negative, thin, rods
5. Colony morphology/growth
- Buffered charcoal yeast extract with cysteine and iron (BCYE) agar plates
- 5 days to grow
- Brown pigment
!
Mycoplasma pneumoniae
!
Characteristics
Lacks a cell wall, poor gram stain (negative), sterols incorporated into cell membrane, aerobic,
extracellular, non-motile, non-encapsulated, and is asporogenous.
!
Humans only reservoir; transmission via respiratory droplets
!
Atypical pneumonia
- Most common cause in college students/military epidemics
- Usually asymptomatic/mild, yet can be fatal
- Unilateral, lower lung involvement; interstitial pneumonia, non-productive cough, fever,
chills headaches, chest pain;
- Symptoms last for weeks, months, relapses occur
- Complications in sickle-cell anemia patients is necrosis of fingers and toes (Raynauds
phenomena, autoagglutinins/antibodies)
!
Diagnostic Tests
1. Carbohydrate fermentation: glucose fermented
2. Tetrazolium dye reduction: blue to yellow
3. Gram stain
- Lacks cell wall and the cell membrane contains sterols. Thus, the result is a poor gram stain
(weak negative?)
- Sputum; takes weeks for culture
!
Neisseria
!
!
i.e. Neisseria meningitides
Characteristics
- Gram-negative diplococci (coffee bean or squashed kidney bean arrangement)
- Aerobic
- Extracellular
- Polysaccharide capsule
- Non-motile
!
Humans are the only reservoir
- Colonize nasopharynx
- Transmission via respiratory droplets
!
1. Epidemic meningitis (children, young adults, military)
- Follows upper respiratory infections, bacteremia
- Fever, neck pain, headache
2. Meningococcemia
- Follows upper respiratory infection
- Rashes of skin, mucous membranes, conjunctiva-> disseminated intravascular coagulation,
(DIC); weakness, hypotension, vascular collapse, shock->rapidly fatal
!
Diagnosis Tests
1. Catalase
2. Oxidase
3. Carbohydrate Utilization: Glucose and Maltose
4. Latex Agglutination
5. PCR
6. Gram stain: gram-negative diplococci
- Coffee bean appearance (adjacent sides slightly flattened)
- Small transparent colonies (usually larger than 1 mm) smooth
- Non-pigmented, nonhemolytic
- Fastidious growth requirements, difficult to culture
- Grows best on chocolate agar or Modified Martin-Thayer medium
!
!
! Pseudomonas aeruginosa
Characteristics
- Gram-negative, long, thin rods
- Flagella; capsule; non-spore forming
- Non-fermentative obligate aerobe
Note: approximately 15% of all gram-negative clinical isolates are “nonfermenters” and of
those, P. aeruginosa represents approximately 70%
Ubiquitous; found in hospital sinks, respiratory equipment, whirlpools, swimming pools, plants,
skin and mucosa
Clinical Presentation
a. Pneumonia (necrotizing bronchopneumonia)
- Nosocomial; immune compromised (cystic fibrosis patients)
- Fever, cough, purulent sputum, cyanosis, lung abscess; lung nodules/cavitation- can be
fatal
b. Burn/wound infections
- Nosocomial; fruity odor with blue-green discoloration, that can lead to necrosis in
adjacent tissue that can become a systemic (fatal) infection
c. Endocarditis (acute/subacute)
- IV drug users
d. Urinary tract infection
- Nosocomial; usually iatrogenic (from catheterization)
e. Bacteremia
- Immune compromised; can seed any system and may be fatal if septic shock develops
f. Corneal keratitis: contact lens-> blindness
g. External otitis (swimmer’s ear)
- Benign to serious; frequently recurrent
Diagnostic Tests
1. Determine OF characteristic of all gram-negative bacilli by inoculate KIA or TSI
2. Oxidase
3. Non-fermenter
4. H2S
5. Oxidase
6. Indole
7. Pigment: blue pyocyanin (water soluble, non-fluorescent)
- Pyoverdins (yellow) plus blue pyocyanin gives green color frequently seen with
P. aeruginosa.
- Red (pyorubin) and brown (pyomelanin) pigments may also be produced
8. Odor is fruity and grapelike (aminoacetophenone)
9. Motility
10. Colony morphology/growth:
- Large colonies; blue-green color; fruity odor. Pigment seen on blood agar plates especially
if > 48 hours. Can grow at 42oC.
11.Gram stain
!
FACULTATIVE ANAEROBES
!
Corynebacterium
!
Corynebacterium diphtheriae
Characteristics
Gram positive irregular bacilli, facultative anaerobes, non-spore forming, non-motile bacteria
!
Humans are the only reservoir (other species found in animals)
- Healthy asymptomatic carriers
!
Diphtheria
- Rare in US; respiratory distress from pseudomembrane/lympadenapathy
- Cardiac toxicity leading to congestive heart failure, neurological toxicity (ie pharyngeal, soft
palate paralysis and peripheral motor neuropathy) caused by bacteriophage encoded exotoxin
!
Diagnostic tests
1. Gram stain and Methylene Blue Stain: smears of nasopharyngeal/throat swab
- Loeffler’s methylene blue stain helpful because do to observation of typical deep blue
staining phosphate or metachromatic granules.
Note: “diptheroid” cells are of various size and shape, range from coccoid to rod shaped,
frequently uneven staining with gram stain. “Snapping” following cell division leads to cell
arrangements described as “picket fence or Chinese figures.” Look for “V, L and Y”
arrangements of cells.
- SBA shows grayish colonies with some strains of C. diptheria are beta-hemolytic.
- Loeffler’s medium (4 to 8 hours) enhances metachromatic granule formation
- Potassium tellurite medium inhibits growth of most of normal flora of upper respiratory
tract, permit corynebactera (C. diphtheriae and saprophytic corynebacteria) to grow,
producing grayish-black colonies.
- Tinsdale medium (contains tellurite) permits differentiation of C. diphtheriae from
commensal type corynebacterium; C. diphtheriae produces brown halo (from H2S released
by cysteinase) around the black colonies.
Note: Staphylococci and Streptococci will grow on tellurite medium forming black colonies but
usually lack the brown halo of C. diphtheriae colonies.
3. Growth on Loefflers Medium
4. Metachromatic staining of high molecular weight polymers of polyhexametaphosphate
storage granules
5. Growth of black colonies on Tellurite Medium
6. Catalase test
Toxigenicity tests
Because beta-phage negative C. diphtheriae may be carried in throat/nasopharynx, definitive
identification of pathogenic strains includes in vivo/in vitro toxigenicity tests. Ten colonies
should be tested as both toxigenic and nontoxigenic variants of a single strain can be isolated
from a patient.
!
a. in vivo: rabbit or guinea pig inoculated intradermally with suspect culture. 5 hours
following C. diphtheriae anti-toxin is given to animal. After 30 minutes, second
intradermal injection of suspect culture is given to the same animal. Necrotic area will
develop at site of first injection, and a pink nodule at the second site, if culture positive
for C. diphtheriae.
b. in vivo: rabbit or guinea pig inoculated intradermally with suspect culture. 5 hours
following C. diphtheriae anti-toxin is given to animal. After 30 minutes, second
intradermal injection of suspect culture is given to the same animal. Necrotic area will
develop at site of first injection, and a pink nodule at the second site, if culture positive
for C. diphtheriae.
!
Enterobacteriaceae
!
Characteristics
Gram-negative bacilli; extracellular, facultative anaerobes; capsules; motile and non-motile;
Salmonella and Shigella are facultative intracellular pathogens. Most common bacteria isolated
from clinical samples
!
Ubiquitous: soil, water, plants, intestinal tracts of humans and animals
!
- Diarrhea and dysentery (E. coli, Salmonella, Shigella)
- Opportunistic pneumonia (Serratia marcescens, Klebsiella, E.coli)
- Urinary tract infections (Proteus, Morganella, Providenicia, Enterobacter, Serratia,
uropathogenic E. coli)
- Neonatal meningitis (uropathogenic E. coli)
- Bacteremia: E. coli; IV drug users are having more infections with Serratia marcescens
- Infective arthritis: Serratia marcescens
- Wound infections (E. coli, Proteus, Klebsiella-Enterobacter group)
!
Diagnostic Tests
1. Oxidase
2. Carbohydrate Fermentation
3. Nitrate Reduction
4. H2s Production
5. Motility
6. IMViC (Indole, Methyl red, Voges-Proskauer, Citrate)
7. Urease
8. TSI
9. Gram stain: short, plump, gram negative bacilli/coccobacilli
a. large, dull gray, dry or mucoid (suggests encapsulated K. pneumoniae) on SBA
b. hemolysis variable and indistinct; (colonies appearing in thin film/waves suggests
motility, probably Proteus)
c. if pink/red colonies on MacConkey or green on EMB (eosin methylene blue) can
form acid from lactose ( lactose fermenters)
!
Note: carbohydrate fermentation = glucose-positive fermentation; lactose fermentation requires
β-galactosidase permease and β-galactosidase. Non-lactose fermenter lack one or both enzymes,
lactose fermenters have both enzymes, late lactose-fermenters have β-galactosidase activity but
“sluggish” β-galactoside permease activity; ONPG test useful to detect late lactose fermenters.
!
! !
E. coli strains
Members the family Enterobacteriaceae: +/- motility; +/- polysaccharide capsule
- Several different types:
o Enterohemorrhagic EHEC
o Enteropathogenic EPEC
o Enterotoxigenic ETEC
o Enteroinvasive EIEC
o Uropathogenic E. coli
!
Shigella species: a member of the family Enterobacteriacea
- Closely related to E. coli
- Gram-negative bacilli
- Non-motile
- Non-encapsulated aerobic, intracellular bacillary dysentery
o Group A: S. dysenteriae types 1-10
o Group B: S. flexneri types 1-6
o Group C: S. boydii types 1-15
o Group D. S. sonnei types 1
!
Human GI tract is reservoir
- Fecal-oral transmission
- Extremely contagious i.e. low infective dose (5x 101 to 3x 102) as extremely resistant to
stomach acid
- Usually seen in young children in group care; may be fatal in elderly or immune compromised
!
- Bacillary Dysentery: large intestinal mucosa invaded without systemic spread; nausea
vomiting, watery or bloody diarrhea; WBC/cell debris in feces. Usually self-limiting.
- Hemolytic Uremic Syndrome: hemolytic anemia, thrombocytopenia, kidney failure.
Note: Shiga toxin = AB toxin. B subunits bind glycoprotein of large intestine to permit A toxin
entry. The cells die due to a disruption of the energy metabolism cycle in the mitochondria.
!
Diagnostic Tests
1. Oxidase 5. Urease
2. Lactose fermentation 6. Ornithine Decarboxylase
3. TSI 7. Motility
4. H2S !
Klebsiella pneumoniae
!
Characteristics
Large, gram-negative rods, aerobic, extracellular; non-motile; large polysaccharide capsule;
member of the family Enterobacteriaceae.
!
- They can be found as part of our normal colonic flora
- Passed via nosocomial transmission by in-dwelling catheters/endotracheal tubes
!
a. Pneumonia: broncho-and lobar pneumonia; thick “currant jelly’ sputum
b. Septicemia: second only to E. coli as cause of nosocomial septicemia
!
Diagnostic Tests
1. Oxidase
2. Carbohydrate Fermentation; Glucose, Lactose and Sucrose Fermented
3. Urease
4. Indole negative ( test to distinguish K. oxytoca and K. pneumoniae)
5. Motility
6. Gram stain: show large, long, gram-negative rods
7. Colony morphology/growth: large, slimy, mucoid, white on blood agar.
- Lactose fermentation on MacConkey agar best seen from the bottom of the plate (lactose
fermenters).
- Non-hemolytic
!
! Salmonella
Characteristics
- Gram-negative bacilli
- Member of the family Enterobacteriaceae
- Facultative intracellular within macrophages but not PMNs
- Facultative anaerobe
- Flagellated/motile
- Polysaccharide capsule
- Somatic O antigen (lipopolysaccharide), protein flagellar H antigens; capsule variable
- S. typhi has capsular (virulence) Vi antigen
!
Note: The most complex members of family Enterobacteriaceae
!
-Fecal-oral transmission, usually via human/animal feces contaminated food, water, milk;
-Yolks of eggs can be colonized from ovarian infected (S. enteriditis) hens
-Requires a large infective dose (105) because most are destroyed by stomach acid
a. non-typhoidal Salmonella
- reservoir is animals
- almost all animals can be infected with non-typhoidal Salmonella (poultry, pet birds,
dogs, cats, horses, cattle, wild terrestrial and aquatic mammals, reptiles, amphibians,
humans)
- children under 5 are susceptible to non-typhoidal Salmonella infections acquired from
pet reptiles (Salmonella serotype arizona, e.g., turtle, iguanas, snakes) with potential
complications of septicemia and meningitis.
b. typhoidal Salmonella
- Humans are reservoir for typhoidal Salmonella but there may be chronic asymptomatic
although human carriers are possible for both
- Humans are the only known reservoirs for S. typhi.
!
Serotype prevalence
- S. typhimurium most frequent isolate (20% of Salmonella isolates)
- S. enteritidis outpaced S. typhimurium in 1989; several outbreaks have been associated with
eggs since 1990
- Estimated that 0.01% of all chicken eggs are S. enteriditis positive
Salmonella classification
- 2200 serotypes
- Groups based on O somatic antigens subdivided into serotypes based on H flagellar antigens
!
a. Gastroenteritis/enterocolitis: S. typhimurium, S. enteritidis, non-typhoidal Salmonella
- Most frequently presents clinically with watery diarrhea, pus in stool, low-grade fever,
nausea, vomiting (can mimic appendicitis)
- Symptoms within 48-hours of ingesting bacteria
- 7-day duration of symptoms but Salmonella may be shed for > than 1 month
b. Bacteremia/septicemia
- Salmonella infecting GI tract, then lymph, thoracic duct and eventually enters the
bloodstream
- High-spiking fever with a positive blood culture
- Can then colonize biliary tract, causing septic arthritis and endocarditis
- AIDS patients may suffer recurrent Salmonella bacteremia
c. Enteric fever: specific type of enteric fever caused by S. typhi or S. paratyphoid
Typhoid fever (S. typhi)
- Classically bimodal
- First phase is 1-2 weeks = positive blood culture
= negative fecal culture
= fever and constipation
- Second phase is about 4 weeks = negative blood culture
= positive fecal culture
= intestinal hemorrhage, delirium
d. Carrier state: especially with S. typhi
- Excrete salmonella in feces for weeks up to a year without clinical symptoms i.e.
(“Typhoid Mary”)
!
Diagnostic Tests
1. Lactose fermentation
2. Oxidase
3. H2S
4. Urease
5. Agglutination reactions: grouping (O antigens), serotyping (H antigens)
6. Gram Stain: gram-negative rod
- Large, gray-white, opaque colonies on blood agar
- Colorless on MacConkeys.
- Can produce H2S, on XLD agar presenting colonies with a black center
- EMB colonies are clear
!
! Vibrio parahaemolyticus
Characteristics
- Gram-negative, “s”-shaped bacilli
- Facultative anaerobe
- Non-encapsulated
- Produce AB toxin
* Requires a large infective dose (1x107) to establish an infection due to its acid-sensitivity
a. V. cholera
- Human only reservoir
- Fecal-oral transmission especially contaminated water
e. V. parahaemolytius
- Free-living in salt water
- Transmitted by eating raw/undercooked seafood or by ingesting food contaminated by
seawater
a. V. cholera
- Non-invasive
- Remains in the lumen of the small intestine
- Painless; odorless “rice-water” diarrhea; rice = mucus
- Afebrile which leads to dehydration, hypotension, and shock—can be fatal within hours
- Abortion
b. V. parahaemolyticus
- Food poisoning = enterotoxin toxic to small intestine mucosal cells; localized invasion of
large intestine (not systemic); within 24 hours of ingestion, abdominal pain, explosive
watery diarrhea
- Self limiting within 3 days.
- Extra-intestinal; otitis externa, wound infections
!
Diagnostic Tests
1. Oxidase
2. Lactose fermentation- weakly acidic by-products (TSI=K/A G- H2S-)
3. Indole production
4. Urease negative
5. TCBS agar:
6. Growth at 7.5% NaCl
7. Production of protoplasts in alkaline peptone water
8. Gram stain: gram negative curved, comma/ S-shaped bacilli
- Growth on blood agar
- Some swarming with smooth, convex creamy grey or white colonies with entire margins
!
!
Yersinia
!
Characteristics
Yersinia was previously described as belonging to the Pasteurella genus, however it is a member
of the family Enterobacteriaceae
- Cells appear small
- Coccibacillary
- Small/pinpoint lac-negative colonies on MacConkey agar
- Optimal growth 25oC to 32oC; motile at 25-28oC
- Considered zoonotic
- Plasmid encoded complement resistance; can grow in cold (4oC)
!
- Animal reservoir, causes epizootics of abortion, diarrhea, pneumonia, and lymphadenopathy
- Asymptomatic blood donors with Y.enterocolitica bacteremia can lead to transfusion reactions
- Proliferates in refrigerated packed erythrocytes stored at 4oC and produces endotoxin after 2-
week lag period.
!
a. Usually self-limiting
b. Transfusion sepsis: shock-like syndrome with chills; follows blood transfusion with
contaminated blood and “cold” enrichment
!
Diagnostic Tests
1. Recovery is low; more biochemically active at room temperature; many labs incubate
fecal culture on MacConkeys at room temperature if Y.enterocolitica is suspected
2. Typical enteric tests; key is temperature requirements
3. Gram stain: small gram negative coccobacilli
- Most strains will grow on selective enteric media
- Small lactose-negative colonies on MacConkey (very slow growing) and SS agar after 48
hours incubation at low temperature or room temperature
- Gray-white convex colonies after 48 hours will appear as a coliform on EMB agar/TSI as
sucrose-positive
Staphylococci
!
Characteristics
- Gram-positive cocci
- Facultative anaerobes
- Non-motile
- Catalase positive
!
Ecology
a. Staphylococcus aureus: anterior nares (nose), skin, oropharynx, and gastrointestinal tract
- 20-60% of adults are carriers
- Disease is rare and occurs when normal bacterial defense mechanisms are overcome or
suppressed.
b. Staphylococcus epidermidis: skin and mucous membranes colonized within few days of birth
- important part of normal flora, that interferes with colonization by more pathogenic bacteria
- disease caused by S. epidermidis usually seen with immune compromised patients and IV
drug users
- Nosocomial/iatrogenic type infections; usually requires trauma to gain access.
c. Staphylococcus saprophyticus: genitourinary skin normal flora
- Infections occur from poor hygiene or sexual activity may lead to urinary tract infections
!
Clinical Presentations
a. Staphylococcus aureus
- skin infection
- bacteremia and sepsis (#1 cause) which can “seed” distant sites and lead to infective
arthritis (#1 cause in adults), osteomyelitis (#1 cause; hematogenous spread or from
trauma), acute endocarditis (#1 cause) normal, abnormal, prosthetic valves, metastatic
abscesses.
- post-viral lobar pneumonia (especially following influenza)
- Toxic Shock Syndrome associated with tampon use
- food poisoning from ingestion of enterotoxin
!
b. Staphylococcus epidermidis
- bacteremia and sepsis (iatrogenic, IV drug users, immnocompromised) may lead to
subacute endocarditis (abnormal or prosthetic valves)
- neonatal bacteremia (especially neonatal ICU)
c. Staphylococcus saprophyticus
- urinary tract infections
- pyelonephritis
- cystitis
- most cases show pyuria
Diagnostic Tests
1. Direct gram-stained smears: gram-positive cocci
- May be in grape like clusters
- Culture and appropriate identification techniques must follow to confirm identification
as Staphylococcus spp.
2. Colony morphology: (usually require >24 hours incubation for species differences to be
noted in colony morphology)
- S. aureus colonies are usually large, 6-8 mm, smooth entire, raised, translucent;
colonies of most strains are pigmented, cream yellow, golden, or matt grey.
- S. epidermidis colonies relatively small, 2.5-6 mm in diameter; usually not pigmented;
slime (glycocalyx) producing strains very sticky, adhere to agar (may be difficult to
determine).
- S. saprophyticus are large colonies, 5-8 mm diameter, very glossy, opaque, smooth,
butyrous, more convex than others; some strains are pigmented, and cream colored.
3. Hemolysis - different hemolytic patterns may be observed around staphylococcal
colonies depending on species, strain and length of incubation
- Complete clearing of blood agar from lysis of RBC = beta-hemolysis
- Caused by alpha toxin (septic shock and dermonecrosis)
- “Shadow”/darkening of blood agar is caused by beta-toxin (“hot-cold hemolysin”) with
sphingomyelinase activity which “sensitizes” the RBC membranes without total lysis
- Incubation at 37o C followed by overnight storage in the cold or will cause the RBCs in
the beta-toxin zone to lyse completely, changing the “shadow” to complete the beta-
hemolysis = “hot-cold hemolysis”
- Beta-toxin of S. aureus is essential for the performance of the CAMP test used in the
speciation of some Streptococci and Listeria.
- S. aureus: at <18-24 hours incubation, may see a “double zone of hemolysis” closest to
colony with a “shadow” of blood agar further away from the colony; may see only beta-
hemolysis; some S. aureus may appear non-hemolytic (<10%)
- S. epidermidis: non-hemolytic (delayed hemolysis >36 hours not caused by alpha-
hemolysin)
- S. saprophyticus: non hemolytic
4. Catalase
5. Coagulase
6. Mannitol fermentation
7. Salt tolerance: growth in the presence of 7.5%NaCl
8. Novobiocin susceptibility to differenciate coagulase negative Staphylococcus
9. Modified oxidase: to distinguish Staphylococcus from Micrococcus
Streptococci
!
Characteristics
- Gram positive cocci
- Usually in pairs or chains
- Most facultative anaerobes
- Non-motile
- Non-spore forming
- Some encapsulated catalase negative
!
Classification
Practical arrangement into major categories based on:
1. Colony morphology and hemolysis (beta, alpha, gamma hemolysis)
2. Serological specificity based on group-specific substance, carbohydrate C or lipoteichoic
acid (Lancefield grouping).
3. Biochemical reactions, resistance to certain factors
4. Ecology
!
Refer to Lancefield grouping (Appendix 7)
!
Clinical presentations
Refer to Lancefield grouping (Appendix 7)
Note: “Infective endocarditis”
a. Acute endocarditis: bacteremia with beta-hemolytic streptococci
- Pneumococci, enterococci, may settle on normal or previously deformed heart valves or
prosthetic valves
- Destruction of valves can lead to cardiac failure within days to weeks
b. Subacute endocarditis
- Frequently involves abnormal valves (congenital, rheumatic fever damaged, atherosclerotic
damaged)
- Thrombi develop on damaged endothelium and act as sites for bacterial colonization
- 30% patients following dental extraction have circulating viridans streptococci which is the
most frequent cause of subacute bacterial endocarditis
- 5-10% subacute endocarditis is from enterococci found in gut/urinary tract
- Slowly progressive disease
- Active inflammation and healing concomitant
- Accumulation of fibrin, platelets, bacteria, blood cells to valves creates “vegetative” lesions
!
Diagnostic Tests
1. Gram stain:
- Gram-positive cocci in chains classically
- Smears from pus often show single cocci or pairs rather than chains, also may appear gram-
negative as cell walls deteriorate
- Smears of throat swabs not helpful as viridans strep always present and appear same as
Group A Streptococcus
!
2. Colony morphology:
- Requires growth in media enriched with blood or tissue fluids, i.e. fastidious
- Colonies 0.5 mm diameter
- Transparent or opaque and domed.
3. Hemolysis on sheep blood agar:
a. beta-hemolysis: Complete lysis of RBCs
- Groups A, B, C (S. equi/dysgalactiae) and G; (group D if rabbit or horse blood used).
- Group A, C and G wide zones of hemolysis, 2 to 4 times diameter of colony;
- Group B zone of hemolysis usually smaller, less obvious
b. alpha hemolysis: Not lysis, changes beta-hemoglobin to met-hemoglobin producing a
greenish-gray or brownish discoloration of agar
- Group G, H,K
- S. pneumonia and viridans streptococci are not assigned a grouping with the Lancefield
system but are alpha-hemolytic
c. gamma hemolysis;
- No effect on RBC (group D)
- Organisms showing no alteration to the SBA, just white colonies usually described as
“nonhemolytic” rather than gamma hemolytic.
- Approximately 11% of Group B are non-hemolytic
Note: Group A Streptococci possess two types of hemolysins, streptolysin S, (SLS, oxygen
stable) and streptolysin O, (SLO, oxygen labile). Most, but not all strains possess both SLS and
SLO. For this reason, anaerobic conditions must be provided to determine hemolytic ability. The
technique used is to streak the surface of a blood agar plate and then stab the blood agar several
times with the inoculating needle to seed cells in the anaerobic bottom layer of agar (see “Special
Plating Techniques- Stab and Streak method).
4. Catalase
5. Bacitracin susceptability- Differenciates GBS and GAS
6. CAMP test- differenciates GBS and GAS
7. Hydrolysis of sodium hippurate- Differenciates GBS and GAS
8. Salt tolerance: growth in the presence of 6.5% NaCl
9. Susceptibility to Optochin-Differenciates viridans and S. pueumoniae
10. Esculin/PYR
11. Serological identification-Lancefield grouping
12. Most clinical labs dealing with human specimens use serological methods for identification
13. Coagglutination: S. aureus coated with specific antiserum reacted with streptococcal antigen
extract
14. Latex agglutination: (polystyrene latex beads carry group specific antisera mixed with
streptococcal extract) have replaced Lancefield extraction capillary precipitin test
15. Group A Streptococcus: enzyme immunoassay (EIA) antigens extracted from throat swab
and reacted with anti-group A antibodies conjugated to enzyme; enzyme substrate is added
and color develops if Group A Strep. antigens are present in throat swab definitive
identification of S. pneumoniae is serological because of 83 different capsular serotypes. The
Quellung test may employ serum pool and type-specific antisera.
!
MICROAEROPHILES
!
Campylobacter
!
Characteristics
A gram-negative, slender, curved, “S”, “gull-winged” or spiral shaped; microaerophilic,
zoonotic, micro-encapsulated, motile; thrives in human bile; non-invasive
!
- Reservoir in domestic animals, poultry especially chickens and turkeys, farm animals
- Transmission usually fecal-oral route via fecal contaminated water, raw milk, food
- Contamination of animal/poultry carcasses at slaughter
- Children can transmit horizontally by fecal-oral route
!
Common infections
- especially in children and young adults; symptoms arise 48 hours post ingestion of bacteria;
watery to bloody diarrhea with pus in feces, abdominal pain, vomiting, fever (can mimic
appendicitis); usually self limiting in approximately 1 week
!
Diagnostic Tests
1. Oxidase Positive
2. Catalase Positive
3. Motility Positive; Cork-screw, Darting Motion
4. Urease Negative
5. Carbohydrate Utilization: neither Ferment Nor Oxidize Carbohydrates
6. Nitrate Reduction Negative
7. Hippurate Hydrolysis Positive
8. Cephalothin Resistant
9. Nalidixic Acid Sensitive
10. Gram Stain: gram-negative spiral curved bacilli, bent or “S”, comma shaped, pairs
resemble seagulls
a. Slow growth on selective media; growth at 37 and 42oC for optimal growth. Selective
“campy” media
b. Microaerophilic; optimal atmosphere: 5% O2, 10% CO2, 85% N2; incubate 42oC with
high humidity
c. Gray, flat, irregular, colonial growth, may “run” along streak line
!
!
!
!
Haemophilus influenzae
!
Characteristics
Gram-negative rod/coccobacilli; microaerophilic; extracellular; nonmotile; polysaccharide
capsule
!
Humans are the only reservoir
- Type b is transmitted via inhalation of respiratory droplets
- Non-typable H. influenzae (nt Hi) is non-encapsulated and normal flora of pharynx and
conjunctiva
!
- Pneumonia (type b and ntHi), especially in young and elderly; fever, cough, purulent sputum
- Meningitis (type b; rarely ntHi): #1 cause in young unvaccinated children 6 mo to 6 years;
fever, neck pain, headache, weakness, nuchal rigidity- can be fatal
- Epiglottitis (strain b): #1 cause especially in young children- can be fatal from airway
obstruction
- Sinusitis/Otitis Media (ntHi): mostly children
- Purulent conjunctivitis (ntHi); can be epidemic; infrequent
!
Diagnostic Tests
1. Catalase-positive
2. Cofactor requirements: V, X factors
3. Serological tests
4. Gram stain: reveals small, gram-negative, pleomorphic bacilli/coccobacilli
- Bipolar staining
- May grow in chains resembling streptococci
5. Colony morphology/growth: colonies are small, translucent, round, “dew drop”, bluish
sheen in oblique light when grown on chocolate agar.
!
Note: Fastidious, Haemophilus requires presence of factor X (which is the heat stable heme,
required for synthesis of respiratory enzymes) and NAD (factor V, the coenzyme nicotinamide
adenine dinucleotide). This is destroyed by autoclaving and by enzymes from unheated red blood
cells). Factor V is inhibited by NAD inhibitors in red blood cells in SBA plates. Both factors can
be provided in chocolate agar (where the heating of the RBCs releases the Factor V), or by
growing on blood agar (providing Factor X) in presence of Staphylococcus aureus which
provides Factor V. Requirements for V/X and type of hemolysis is helpful in speciation.
!
!
! Helicobacter
Characteristics
Small, gram-negative bacilli in “u” or “gull-wing” “s” that are comma shaped; microaerophilic,
extracellular; amphitrichous flagella, “corkscrew” movement; non-encapsulated; urease and
mucinase permit life in hostile stomachs; non-invasive bacteria.
!
- Fecal-oral transmission; increased prevalence with age
!
a. Stomach/lumen; non-invasive
b. Chronic gastritis (type B) #1 cause; non-invasive; superficial mucosal inflammation
c. Duodenal peptic ulcer: #1 cause
d. Gastric peptic ulcer: #1 cause
e. Gastric carcinoma
Note: regarding the clinical presentations
- Deep-seated abscesses, necrotizing lesions; animal bites, food-borne botulism, septic
abortion, abscesses of any organ, surgical complications.
- Work in concert with other bacteria with trauma, vascular stasis or tissue necrosis to lower
the oxygen tension to establish favorable conditions for growth of anaerobes.
- Polymicrobial with non-pathogenic bacteria results in synergy to increase severity of the
infectious process.
!
Diagnostic Tests
1. Rapid urease activity
2. Oxidase positive
3. Catalase positive
4. Nalidixic acid resistant; cephalothin sensitive
5. H2S negative
6. Gram stain: gram negative bacilli, “u” or “gull-wing” shaped.
7. Colony morphology/growth: selective media used is Brucella-heart infusion; same
atmosphere as for Campylobacter.
!
Transmission
1. Exogenous infections (outside sources)
- Contaminated food
- Nosocomial infections
- Wound infections (contaminated by soil or other outside sources)
- Infections following bites
2. Endogenous infections (infections from host’s normal flora)
- Surgery trauma with metastatic spread
- Brain abscesses, endocarditis, spinal osteomyelitis
- Necrosis of membrane tissue as in ischemic bowel syndrome
- Immunosuppressive drug therapy- compromised host resistance-necessary
- Inoculation of oral flora into lungs by aspiration, or with iv drug users who use saliva as a
diluent
ACID FAST ORGANISMS
!
Mycobacterium
!
Characteristics
Acid-fast bacilli, non-motile, non-spore forming, facultative intracellular pathogens
!
Ecology
1. Mycobacterium tuberculosis
- the main reservoir is humans
- transmitted principally by infected aerosols/ droplet-nuclei
- can be transmitted from people to animals (i.e. animal caretakers with active pulmonary
tuberculosis)
!
2. Mycobacterium bovis
- The main host is cattle and the bacteria is excreted in the milk
- Humans acquire M. bovis via the gastrointestinal tract by drinking infected, unpasteurized
- Milk.
!
Diagnostic tests
1. Clinical: chest x-ray; dense infiltrates of lung/solitary nodules with well-defined margins
2. PPD skin test positive
3. Acid Fast Stain
4. Gram stain/acid fast stain
a. may stain weakly gram-positive
- M. tuberculosis in clinical smears = relatively short, thin, beaded, slightly curved, red-
staining, acid-fast bacilli.
- From broth, acid-fast bacilli are seen arranged in parallel sheaths highly suggestive of
M. tuberculosis; aggregates due to production of cording factor (virulence factor).
b. Acid fast stains- stains bind to mycolic acids in cell wall
5. Specimens
- The specimen is a sputum sample, early morning for 3-5 days; urine; feces; blood; CSF;
tissue biopsy; and/or needle aspirates.
- The specimens are frequently treated with strong acid or alkali to decrease undesirable
contaminating bacteria and to liquefy mucus; the high concentration of lipids in cell walls
of mycobacterium makes them more resistant to killing by these methods.
6. Pigment *M. tuberculosis will not produce pigment even after exposed to light as with other
Mycobacteria
7. Colony Morphology/growth
- M. tuberculosis: non-pigmented, rough, buff colonies in 14-28 days at 37oC on
Lowenstein Jensen or Middlebrook media
8. Catalase
9. Generate Niacin
10. Pigment
11. Nitrate Reduction
12. Gas-liquid chromatography/high-performance liquid chromatography (GLC/HPLC)
determines the composition of cell wall mycolic acids and fatty acids help in rapid species
identification of mycobacteria from recovered clinical samples.
13. Nonisotopic nucleic acid probes: specific probes available for culture confirmation.
14. PCR of mycobacteria DNA from clinical samples i.e. sputum
!
* Additional tests for M. leprae*
15. Lepromin skin test
16. Cannot culture in vitro (grow in foot pad of mouse or nine-banded armadillo)
17. Serology
18. Stain of biopsy
19. dopa-oxidase
!
Note: Slow growing mycobacteria takes weeks to diagnose using conventional methods.
Research is continuing develop molecular techniques to decrease time of diagnosis and to
determine antibiotic susceptibility of multi-drug resistant strains.
!
Nocardia
!
Characteristics
Aerobic, gram-positive bacteria which are often branched and filamentous. Mycelium may be
produced which breaks into rod-shaped/coccoid forms. Frequently are worked up in mycology
but these are not fungi; partially acid-fast
!
a. Soil, compost, decaying vegetation; inhalation of airborne “conidia” leads to pneumonitis,
dissemination.
b. Opportunist pathogen of the immune compromised.
- May occasionally be saprophytic /transient member of respiratory tract flora.
- Can grow inside macrophages and produce catalase and superoxide dismutase which
counters the effects of macrophage myeloperoxidase system.
- T-cell immunity important.
!
a. pulmonary abscess (cavitating lesions; confused with TB)
b. sepsis (spread to any organ system; brain abscesses develop in 30% of disseminated cases)
c. Mycetoma (chronic granuloma subcutaneous tissue; draining sinus tract)
- May be caused by subcutaneous implantation of any of the nocardia/aerobic
- Actinomyces.
- Suppurating abscesses, draining tracts, granulomas.
- White or yellow grains and granules, 1 mm in diameter in purulent material are often
observed.
!
Diagnostic tests
1. Modified Ziehl-Neelson stain of sputum:
- Nocardia appears partially acid-fast
- Thin, delicate beaded or branched filamentous red staining rods
Note: Smears of Nocardia grown in broth more frequently demonstrate branched filaments.
2. Gram stain biopsy
3. Culture
4. Catalase test
5. Pigment-usually a shade of yellow
6. Colony morphology/growth:
- Can grow on sheep blood agar (SBA), BHI agar, and Sabouraud dextrose agar
- Nocardia can grow in a 42-45oC (inhibits growth of other bacteria) and can take 4 to 6
weeks to grow
- 10% CO2 enhances growth.
- colonies typically dry, chalky, heaped or folded;
- Odor like newly turned soil or musty basement.
- Temperature range is 30-36oC in CO2 or ambient air.
- Grows well on mycobacteria media such as Middlebrook (glabrous, wavy, adherent
colonies) or Lowenstein-Jensen or fungal media.
NON-SPORE FORMING GRAM NEGATIVE PLEOMORPHICS
!
A. Bacteroides fragilis group (obligate anaerobes)
- The clinical specimen is a pale, irregular, pleomorphic, gram-negative rod with bipolar
staining in a smear from an abscess
- Non-motile, gram negative, pleomorphic, polysaccharide capsule
- Part of the indigenous microbiota of the intestinal tract, but is less than 1% of normal
flora
- Colonies are 1-4 mm in diameter, opaque with concentric whorls and ring-like structures
- Generally cause endogenous infections
!
Diagnostic Tests
1. growth Enhanced by Bile
2. hydrolyze Esculin
3. resistant to Penicillin, Kanamycin and Vancomycin
4. saccharolytic
!
B. Prevotella intermedia
- Non-motile, gram-negative coccobacilli
- Pigmented (2 days to 3 weeks to produce)
- Small-medium convex, entire edge, hemolytic
- Brick-red fluoresce under UV light
- Part of the normal flora of the oropharynx, nose, GI and GU tract.
- Isolated from the oral cavity
- Infections involving head, neck, lower respiratory tract, and urogenital tract
!
Diagnostic Tests
1. saccharolytic
2. indole positive
3. esculin, ribose hydrolysis negative
!
C. Fusobacterium nucleatum
- A clinical specimen is pale, irregular, pleomorphic, gram-negative rods with bipolar
staining in a smear from an abscess
- Gram-negative, long, filamentous with tapered ends, varies in length
- Colonies are slightly convex with irregular margins with an internal flecking
- Normal flora of the GI and GU tract and upper respiratory
- Pleuropulmonary infections, brain abscesses, chronic sinusitis, metastatic, osteomyelitis,
septic arthritis, liver abscesses
!
Diagnostic Tests
1. Produces butyric acid
2. Biochemically inactive
!
NON-SPORE FORMING GRAM POSITIVE COCCOBACILLI,
PLEOMORPHIC
!
A. Propionibacterium acnes
- Gram-positive, “diphtheriod-pleomorphic”
- Colonies are circular, convex, glistening, and opaque
- Some strains have a narrow zone of hemolysis and grow microareophilicaly
- Normal flora of the skin, nasopharynx, oral cavity, GI and GU tracts
- Catalase negative
- Indole negative
!
B. Actinomyces
- Gram-positive, thin, and branched filaments that vary in length
- Rough granular or spider colonies
- 7-10 day old colonies- raised and heaped, irregular or lobate margins = molar tooth
- Normal flora of the mouth, and perhaps the GI tract
- 60% of infections are cervicofacial; chronic localized, the rest in abdominal (especially
appendix and thoracic region)
!
Diagnostic Tests
1. Sulfur granules showing peripheral clubs with branched filamentous rods from a
cervicofacial tissue exudate- macroscopically visible also
2. Ferments xylose, mannitol
3. Reduces nitrate
4. Does not hydrolyze starch
5. Catalase negative
6. Culture lesion granules on BHI 2% agar anaerobically 4-6 days
!
! PATHOGENIC FUNGI
!
Dermatophytes
Characteristics
- A superficial mycoses of the keratinized epidermis and epidermal appendages (i.e. hair, nails)
producing a mild but chronic infection
- Causes tinea (ringworm) - expands in a circular pattern in or on hair, in or on nails
- All strains can utilize keratin. some produce keratinase and proteolytic enzymes that induce
intense inflammation
- Necessary to identify by microscopic examination of KOH treated specimen to eliminate
psoriasis or contact dermatitis as a possible cause of disease. If KOH examination is negative,
specimens are cultured.
A. Microsporum gypseum
- Colony morphology is characterized by agranular surface to a buff colony which becomes
cottony after 2-4 weeks.
- Sabouraud’s medium produces a roughened, thick-walled marcoconidia, few or no
- Microconidia
- Macroconidia is less barrel-shaped with rounded tips and more numerous
- Invades skin and hair inside and outside the hair shafts causing the hair to break above the
shaft
B. Trychophyton mentagrophytes
- Colony formation varies from smooth to powdery, white, pink, red or purple many
microconidia, that may form grape-like clusters
- Few or no macroconidia that are thin-walled, pencil-shaped and multicellular
- Grows on hair, skin or nails
Ecology/ Transmission
- Found in soil
- Can infect animals
Note: tinea = ring-like lesion
1. Tinea capitas -scalp area. There are many forms depending on the causative agent that
may or may not invade the hair shaft, or cause the hair to break at the skin.
2. Tinea corporis- body. Annular lesions on the smooth skin that have a spreading
hemorrhagic border
3. Tinea barbae- beared area. Zoophilic inflammatory lesions many from T.
mentagrophytes, found in farm workers
4. Tinea cruris- groin. “Jock rot” Inflammatory with enlarging margins. Infectious and
found with athletes and ship crews who share the same towels and linens.
5. Tinea pedis- feet. Referred to as “athlete’s foot”, itchy, scaly or seeping lesions. Most
common during the warm and humid months.
6. Tinea unguium-nails. The nails become thickened and brittle.
Diagnostic Tests
1. Clinical presentation via location and type of lesions
2. Micro-morphologic differences from other pathogenic fungi
3. Spore type
4. Response to antimycotic drugs
PATHOGENIC YEASTS
!
Candida albicans
!
Characteristics
- Unicellular, nucleated yeast-type organism which reproduces by budding
- Fermenter
- Candidiasis is the most common of the systemic mycoses and can present a wide spectrum of
clinical states initiated by a breakdown in the normal host defense system.
!
Normal flora of humans that colonizes in the mucous membranes shortly after birth
- Normal flora of many animals
- Endogenous infections
- Colonization is demonstrated by the appearance of psuedohyphae or hyphae.
- Alterations in the normal flora from prolonged use of antibiotics may predispose to
- Infection
- Low pH of salivary secretions
!
a. often the initial signs with AIDS or other immune compromised conditions where the
infection can become systemic
b. hypothyroidism, diabetes or other endocrine abnormalities predispose individuals to
infections of the skin and mucosa
c. cutaneous infections occur as a direct result of abrasions or continued excess moisture
d. oral candidiasis (thrush) is the most common. The infections are manifested by white
patches on the buccal mucosa and tongue which may coalesce into a membrane in more
serious infections.
!
Diagnostic tests
1. Prompt culture and examination before the specimen is “over grown” by normal flora.
2. Skin scrapings of exposed lesions; treated with 10%KOH to dissolve contaminating skin
or hair will show budding yeast cells and psuedohyphae
3. Cells from deep lesions obtained aspiration (to avoid normal skin contamination) and
stained with Lactophenol Cotton Blue
4. Positive germ tube test
5. Growth on cornmeal agar to identify morphology.
- hyphae
- distinct points of constriction simulating link sausages (pseudohyphae)
- with budding yeast forms (blastospores)
6. Urease negative
7. Growth at 370
Cryptococcus neoformans
!
Characteristics
Gram Stain:
- Irregular sized spherical yeast cells 4-10um in diameter
- Demonstration of a clear well-demarcated halo or capsule with India ink stain
Culture/Colony Morphology:
- Colonies are flat, shining sticky mucoid with smooth edges
- Cream colored becoming tannish, sticky, mucoid colony when grown on blood agar
- The capsule can be demonstrated by slowly extracting an inoculating needle from the
surface
!
Ecology/ Transmission
- Opportunistic infections with lungs as the portal of entry
- Found in soil where the pH is alkaline and nitrogen rich
- Concentrated in the feces of birds such as turkeys, pigeons and starlings.
- Poultry farm workers and city park workers are at a higher risk
!
a. May not become manifest until it migrates to other areas of the body besides the lungs
b. CNS involvement begins insidiously and will manifest to more significant
symptoms such as meningitis, hemiparesis, increased intracranial pressure.
c. May remain subclinical, but clinical disease can be fatal if left untreated
!
Diagnostic Tests
1. The recovery of C. neoformans from clinical specimens is considered significant
2. Grows at 370
3. No pseudo hyphae formed when grown of cornmeal agar, yeast forms generally with a
thick polysaccharide capsule best seen with the India ink stain
4. Urease positive
5. Germ tube test negative
!
!
DEFINITIONS OF MORPHOLOGY
!
1. Annelid a cell that produces and extrudes conidia. The tip tapers, lengthens and acquires
a ring as each conidium is released.
2. Arthroconidium an asexual spore formed by breaking up of a hypha at the point of
septation
3. Arthrospores the segments from mycelium which break apart, each is capable of forming
a hypha and a new mycelium
4. Blastoconidium a conidium formed by budding along the hypha or psuedohyphae
5. Blastospores yeast structure formed by budding
6. Budding a process of asexual reproduction in which the new cell develops as a smaller
outgrowth from the parent cell
7. Chlamydospore thick-walled vesicle formed by C. albicans. It does not germinate or
produce conidia
8. Conidiophore a special hyphal structure that serves as a stalk on which conidia are
formed. The shape and arrangement are characteristic of the genus
9. Conidium asexual propagule (a unit that can give rise to another organism) that forms on
the side or end of the hypha or conidiophore (not within a sac). Single celled are
microconidia and longer, multi-celled are marcoconidia
10. Hypae long, tubular, threadlike structure branched and extended at the tips. Many
together form the mycelium
11. Mycelium several hyphae forming a intertwined filamentous psuedohyphae a chain of
cells formed by budding. Differs from a true hypha by being constricted at the septa and
having terminal cells smaller than the other cells.
12. Septate hyphae that are divided into individual cells by transverse walls that form the
boundaries of individual mononucleotide cells
13. Sporangiophore a specialized hyphal branch bearing a sporangium
14. Sporangium a closed sac-like structure in which asexual spores are formed by cleavage.
!
SPIROCHETES
!
!
Characteristics
- flexible, helical-shaped, poorly gram-staining bacteria
- viewed by dark field
- rotating and flexing type of motility even in viscous media and along a solid surface
- 0.09-0.75 um diameters and 3-500 um long
- non-pigmented
- some are obligate aerobes; some are obligate anaerobes
- diverse characteristics within genus; various nutritional requirements, complex to simple
media; some cannot be cultured
- cellular components are referred to as the protoplasmic cylinder between the outer envelope
and the cytoplasmic membrane are the flagella, axial filaments, which are attached bipolarly in
a subterminal position and extend toward the opposite end of the cell these are structurally and
chemically similar to the flagella of other bacteria
Pathogenic spirochetes
- may also live in the soil and water
- infect domestic and livestock animals
- humans are a dead end with infections as transmission from human to human is very rare
- transmission via breaks in the skin; vectors such as ticks and fleas; or exposure to
contaminated water, soil
- Borrelia: a spirochete, 18-30 µm long and 0.2 - 0.3 µm in diameter. Does not gram stain well,
best visualized by dark field microscopy. (Please review general information about spirochetes
(page 61))
!
- Vector-born (tick); various hosts, deer, dog, white footed mouse, birds
- Humans are a dead-end host
- Infection from tick adult or nymph bites
- Seasonal variation that follows the life cycle of the tick
- Most prevalent in the northeast, Northwest and Midwest
!
Borrelia
a. Lyme Disease (B. burgdorferi)
- Vector is the tick Ixodes ricinus complex (I. dammini in the eastern and midwest and I.
pacificus in the west coast)
- Skin lesion (erythema migrans) 67%-83%; spreads outward; may have central clearing
- 3-32 day incubation; flu-like symptoms
- Cardiac involvement in 8-10%
- Musculoskeletal manifestations, intermittent arthritis and chronic erosive synovitis- 50-
60%
- Bell’s Palsy-cranial neuropathy
b. Relapsing Fever: louse born (human body louse; must go through human to maintain
infectivity
- B. recurrentis; average attack is 5 days with an interval of about 9 days; abrupt onset
with high fever, chills, severe muscle and joint aches
- Tick born (soft body tick Ornithidorus; B. hermsii; depends of the habits of the local
vector-dead trees, rodents; hunter’s cabins; clinical symptoms same as louse born
Leptospira
- Pathogenic-complex based on antigenic characteristics and subdivided into serovars
- Broad host range
- Can reside and multiply in the kidney tubules and shed in the urine
- Contaminates water supply (can survive > 3 months)
- Antibodies are leptospiricidal except in the kidney, eyes and brain where the bacteria
will persist
- Leptospirosis - duration of infection depends on the host and serovar
Treponema
- Pathogenic; T. pallidum = syphilis
- Non-venereal; diseases - T. carateum = “pinta”; T. pertenue = “yaws”
- Morphologically indistinguishable
- Cannot be cultured
- Oral treponemes live in the gingival crevice; excessive accumulation of plaque with
increase number of spirochetes can cause periodontal disease
Diagnostic Tests
Borrelia
1. clinical presentation
2. history of travel or exposure to endemic areas
3. culture (very slow growing)
4. blood smears stained with acid aniline dyes (Wright’s or Geimsa’s) during febrile stage
5. IFA
6. ELISA
7. Western immunoblot with positive reactivity to specific B. burgdorferi outer surface
proteins
Leptospira
Demonstration of the bacteria by culture- slow growing- up to 30 days therefore not generally
used as a method of diagnosis PCR; primers are used to amplify a DNA fragment specific for
Leptospira in tissue or specimens
MAT: Mixing live fluid culture with serum dilutions; after 3 hour incubation, white spheres are
seen under microscopic examination
Treponema
Demonstration of the bacteria by culture (T. pallidum by inoculation rabbit testes); direct
detection of spirochetes from genital lesions
- T. pallidum; VDRL (reference non-treponemal antibody test NTAT), forms microscopic
clumps; positive is a past or present infection; increase predictive value when combine with
the fluorescent antibody absorption test. If NTAT is positive must do Treponemal antigen test
using live Treponema pallidum. Maintained and harvested from rabbit testis.
TOOTH DECAY AND PERIODONTAL DISEASE
!
There is a natural repulsion between the tooth surface and the bacteria found in the oral
cavity. The primary defense mechanism against bacterial colonization is the acidic
glycoproteins (mucins) that are absorbed from saliva by the enamel surface of the tooth. It
forms a membranous layer call acquired enamel pellicle. Because this pellicle contains many
sulfate and carboxylate groups, it has a net negative charge as do the bacteria creating an
inherent, natural repulsion. This defense mechanism breaks down when dental plaque forms.
Plaque forms from the colonization of the pellicle by S. gordonii, S. oralis and S. mitis.
Once the tooth is colonized, then co-aggregation of other bacteria occurs, especially by
Actinomyces viscosus and S. mutans. The streptococci produce extracellular enzymes that
polymerize glucose into glucan polymers. These bind the bacterial cells together. A low
oxidation reduction potential is formed on the surface of the tooth. Now, colonization occurs
with strict anaerobic bacteria, especially between teeth and gums and teeth.
The bacteria produce lactic acid and acetic acids from the sucrose and other sugars that
they metabolize. Because the plaque is not permeable to saliva, none of the acids are
neutralized or diluted so they are able to demineralize the enamel to produce a lesion on the
tooth.
Peridontal disease is a group of diseases that affects the periodontium or the
supporting structure of a tooth. This includes the cementum or the peridontal membrane of the
bones of the jaw and the gingivae. The gingiva is the tissue covered by a mucous membrane
that surround the neck of the tooth and covers the jaw. Disease begins with the formation of
plaque where the tooth and this tissue meet. Porphyromonas gingivalis has been show to be
the main species responsible for the breakdown of tissue through the excretion of a trypsin-
like protease. Additionally, there is an initial inflammatory reaction which induces swelling
and the formation of peridental pockets. Bacteria colonize these pockets, causing more
inflammation, an abscess formation, bone destruction and tissue necrosis.
!
Appendices
Appendices page+#
Appendix(1(*(Stains(and(staining(protocol
(*(Gram(stain 2
(*(India(ink(stain 2
(*(Lactophenol(cotton(blue 2
(*(Loeffler(methylene(blue(stain 2
Appendix(2(*(Miscellaneous(tests(and(reagents
(*(API(test 3
(*(Antibiotic(susceptibility(test 3
(*(Catalase(test 3
(*(Indole(spot(test 4
(*(3%(KOH 4
(*(McFarland(standard 4
(*(Oxidase 4
(*(Satellite(plate 5
(*(X(and(V(factors 5
Appendix(3(*(Selective(and(non*selective(plating(media 6*7
Appendix(4(*(Tube(media,(broths(and(agar(slants 8*11
Appendix(5(*(Media(recipes 12*18
Appendix(6(*(Antibiotic(disk(diffusion(assay 19*20
Appendix(7(*(Lancefield(grouping(for(Streptococcus 21*22
!
APPENDIX 1
!
!
! STAINS AND STAINING PROTOCOL
GRAM STAIN
Crystal violet is the primary stain that binds to the bacterial cell wall in conjunction with iodine.
Bacteria which have the capability to retain the stain after treatment with a decolorizer (alcohol-
acetone), appear dark blue/purple and are referred to as gram positive. Others loose the color but
pick up the counterstain of safranin and appear red. Older gram positive bacteria may also appear
red, as they loose the ability to retain the primary stain with age.
METHOD
Smears are air dried and heat fixed by gently passing through a flame.
Flood slide with crystal violet and let sit one minute.
Add iodine solution and let sit one minute.
De-stain with acetone/alcohol
Rinse in tap water and repeat the de-stain procedure and tap water rinse
Flood slide with safranin and leave for one minute.
Rinse under tap water and blot dry to examine.
!
INDIA INK STAIN
Used in the detection of the capsule of Cryptococcus neoformans.
METHOD
Place a drop of India Ink on a microscope slide
Place a drop of dI water a centimeter away from the India Ink
Gently touch an isolated colony and mix carefully in the dI water and drag the emulsion
to the edge of the drop of India Ink
Add a cover slip to both drops, where the stain appears light grey, forming a gradation of
stain
Where the two meet look for a large white capsule, with the yeast cell in the center.
!
LACTOPHENOL COTTON BLUE
This dye is also a mounting fluid for yeasts and fungi. The lactophenol acts as a mounting
media by rapidly killing the cells without lysing due to the inactivation of lytic cellular enzymes.
The cotton blue is an acid dye that stains chitin and cellulose.
METHOD
The Difco dispensers are squeezed to break the glass ampule.
Add a drop to a slide and with a sterile loop, mix a small sample of culture.
Place a cover slip to examine.
!
LOEFFLER METHYLENE BLUE STAIN
Metachromatic granules of C. diphtheria readily take up the dye and appear deep blue.
Poorly staining gram negative bacteria are better visualized with this stain, especially in
cerebrospinal fluid specimens. Thus dye is also used in the Quellung test.
METHOD
Flood air dried, heat fixed slide with the stain for one minute.
Rinse with water and blot dry to examine
! Appendices!–!page!2!
APPENDIX 2
MISCELLANEOUS TESTS AND REAGENTS
API TEST
This test is made up of various cupules containing dehydrated selective media that test for
different metabolic abilities under aerobic and anaerobic conditions. API strips were developed
to facilitate the identification of bacteria for researchers working in areas where well-stocked
labs were not available. Many different API tests are available depending on the type of
organism (fecal organisms, Streptococcus spp., yeasts, etc.).
METHOD
Depending on the organism, emulsify the appropriate amount of culture in the provided
tubes (saline, dI, etc.), making sure to establish uniform turbidity
Pipette the emulsion into each cupule, filling the caps only when indicated
Apply mineral oil overlay where indicated
Incubate for 24 hours and add the appropriate reagents
Score the API with the paper provided and identify your organism
!
!
ANTIBIOTIC SUSCEPTIBILITY DISCS
Various organisms can be identified by their susceptibility or resistance to particular antibiotics.
Commercial filter discs are saturated with the antibiotic at known concentrations. These are not
used for treatment nor in the antibiotic diffusion assay but solely as a means of differentiation.
METHOD
Streak a SBA plate in four quadrants for isolation; or a lawn (about the size of a silver
dollar) is made on a SBA plate.
With an ETOH flamed forceps, place the disc in the center of quadrant 1 or the center of
the lawn and incubate the plate for 24 hours.
Sensitive: There will be a zone of no growth ≥ a specific diameter around the disc
Resistant: Growth will occur up to and under the disc.
!
CATALASE
Catalase, which is present in most cytochrome-containing aerobic and facultative anaerobic
bacteria. (The main exception is Streptococcus spp). It decomposes the hydrogen peroxide that
can be toxic to the bacteria. Catalase also inactivates free radicals formed by the
myeloperoxidase system within phagocytic cells after they ingest the bacteria.
H2O2 ↔ H2O+O2
METHOD
Pick the center of an isolated colony and place on a clean glass slide.
Add a drop of 3% H2O2
Do not mix
Positive: Vigorous bubbling
Negative: No effect upon addition of catalase or minimal bubbles appear after some time
NOTE: Any blood product carry-over can result in a weak positive reaction from the peroxidase
in erythrocytes.
INDOLE SPOT TEST
A rapid method used in the identification of Enterbacteriaceae and other Gram negative
bacilli that tests for the presence of the enzyme tryptophanase. Tryptophanase hydrolyzes
tryptophan to produce three end products, one of which is indole. The reagents in the indole spot
test include aldehydes which react with the indole end product, producing the positive color
change.
METHOD
Lightly saturate filter paper with the commercial reagent. With a sterilized loop, pick an
isolated colony and rub into the reagent.
Positive: Immediate blue/purple color.
Negative: pink or no color
!
3% KOH
A definitive test for gram negative bacteria. Older colonies or a poor staining technique may give
ambiguous results. The solution will lyse gram negative bacteria, releasing the DNA and forming
the gooey consistency seen with a positive result. Gram positive bacteria will not be lysed.
METHOD
Place an isolated colony on a clean microscope slide.
Add a drop of 3% KOH and mix
Positive: Slowly withdraw the loop and look for stringy substance that adheres to the loop and
the emulsion
Negative: No change in texture of emulsion
!
MCFARLAND STANDARD
A standard used to measure bacterial turbidity. Can be made to approximate various bacterial
concentrations. A ratio of 1% BaCl2 and 1% H2SO4 determines the standards.
Example: 0.5 MacFarland standard used for the Kirby Bauer Assay
50ul of 1% BaCl2 and 9.95ml 1% H2SO4
Equals 1.5x108 cells/ml
METHOD:
Used to equate a suspension of organism to a 0.5 MacFarland standard.
Emulsify an isolated colony of the bacteria in 1.0ml normal saline in the same type of
tube as the standard. Add additional sterile saline to match the turbidity of the standard.
!
!
OXIDASE
A quantitative procedure for determining the presence of cytochrome oxidase activity. This is an
intracellular system that catalyzes the oxidation of cytochrome c by molecular oxygen which
serves as the terminal electron acceptor. Samples should be taken from 24 hour cultures for
accurate results.
METHOD
Carefully remove an oxidase strip from its container
Using a sterilized loop, take a sample of a 24 hour colony and smear onto the strip
(for a better effect, smear a thin layer of culture onto the strip)
Positive: Dark blue/indigo color within 10 seconds
Negative: No color change or appearance of dark blue color after an extended period of time
SATELLITE PLATE
A method for demonstration of Haemophilus on SBA. Although heme (X factor) is readily
available on SBA plates, Haemophilus is unable to grow independently due to its inability to lyse
the red blood cells to gain access to the NAD (V factor) in the cells. For Haemophilus to grow on
SBA, it must be plated with beta-hemolytic bacteria, most commonly S.aureus. S.aureus is able
to lyse the RBC and release the V factor for Haemophilus. Since NAD will only be released in
the zones of hemolysis, Haemophilus will only be able to grow in those areas, creating rings of
colonies around S.aureus, resulting in the satellite morphology.
METHOD
Either by streaking or swabbing, create a lawn of Haemophilus on SBA
Streak S.aureus for isolation on top of the lawn
Incubate the plate for 24 hours in microaerophilic conditions
Examine the plate the next day for growth of Haemophilus around S.aureus colonies
!
X AND V FACTORS
A method for demonstration of Haemophilus’ nutritional needs on BHI agar. Haemophilus have
specific nutritional requirements and cannot thrive without a source of NAD (V factor) and heat
stable heme (X factor) in their environment. SBA and Chocolate agar provide the X factor and
both factors, respectively, so BHI agar is needed for this demonstration.
METHOD
Either by streaking or swabbing, create a lawn of Haemphilus on BHI agar
Quarter the agar plate into four equal sections
Quadrant 1 will be the negative control—no disk will be placed on the agar
In Quadrant 2, aseptically place a V factor disk in the middle of the quadrant
In Quadrant 3, aseptically place an X and V factor disk in the middle of the quadrant
In Quadrant 4, aseptically place an X factor disk in the middle of the quadrant
Incubate the plate microaerophilically for 24 hours at 37ºC
The V factor quadrant must ALWAYS be opposite the X factor quadrant. This is to minimize the
diffusion of the factors into the each others’ quadrant, creating a false reading of the test.
!
INTERPRETATION
There should be no growth in Quadrants 1, 2 and 4. Growth in Quadrant 3 should only be present
as a ring around the disk. The density of growth should be proportional to the proximity of the
colonies to the disk with the densest areas closest to the disk and growth thinning out as we move
away from the disk (as the concentration of X and V factor diffused into the agar decreases).
APPENDIX 3
!
SELECTIVE AND NON-SELECTIVE PLATING MEDIA
!
!
!
BRAIN HEART INFUSION AGAR
Nutrient rich agar used whenever a bloodless medium is needed, such as cultivating the
yeast phase of dimorphic fungi, or identifying the X and V factor needed for Haemophilus
species. Can also be used for clinical isolation of Nocardia asteroides.
!
CAMPY-CVA AGAR
Highly selective medium for the isolation of Campylobacter from clinical samples.
Brucella base agar is supplemented with vancomycin, trimethoprin, polymixin B, cephalothin
and amphotericin. The antibiotics will inhibit any normal flora contaminate.
!
CHOCOLATE AGAR
Isolation and supportive medium for fastidious bacteria such as Neisseria and
Haemophilus. Sheep blood is added to a rich base media and lysed by heating. The hemin (X
factor) and porphyrin (V factor) are then released into the media. The high temperatures used in
the preparation also inactivate the enzyme that breaks down V factor, normally present in sheep
blood.
!
CORN MEAL AGAR WITH TWEEN 80
A type of media used in selecting fungal growth; serves the same purpose as the potato
flake agar, but also allows the formation of chlamydospores in Candida albicans which does not
occur with Cryptococcus spp. Something in the agar induces the formation of C.albicans
chlamydospores by doing something. This media is also used in producing samples for staining
with lactophenol cotton blue. (See Lactophenol Cotton Blue Stain in Appendix 1)
METHOD (for preparation for lactophenol cotton blue staining)
Streak sample onto agar for isolation
Aseptically place a piece of clear tape between Quadrants 1 and 2
After incubation, carefully lift tape and place on a clean slide
Follow instructions for staining
!
EGG YOLK AGAR
Selective and demonstrative medium for Clostridia. The presence of lecithinase is shown
by the appearance of opaque zones around colonies (degraded by-products of lecithin). Lipase is
shown by an iridescent sheen on the colony surface
!
MACCONKEY AGAR
Selective medium for the isolation of enterics or other organisms. Also inhibits the
swarming of Proteus due to higher agar concentration. Bile salts and crystal violet inhibit the
growth of gram positive bacteria and some fastidious gram negative bacteria. Lactose is the sole
carbohydrate so lactose fermenters turn brick red from the absorption and conversion of neutral
red caused by the acid end products. Non-lactose fermenters produce clear colonies.
MANNITOL-SALT AGAR
Selective medium for the isolation of S.aureus from clinical samples. The 7.5% sodium
chloride discourages the growth of all other organisms (except Enterococcus). There is a yellow
zone around the isolated colonies, which is the acid end-product from the mannitol fermentation.
Rarely other mannitol-positive organisms may be recovered on this medium, so it is necessary to
test for the presence of coagulase as further identification. It is not always necessary to streak for
isolated colonies with media. If the determination is the ability to ferment mannitol or grow in
the presence of high salt, and the suspected is taken from a pure culture, then it is not necessary
to streak for isolated colonies.
!
MYP AGAR
Selective medium for the isolation of Bacillus cereus from food. Lecithin in the agar is
degraded by lecithinase, appearing as opaque zones around lecithinase + colonies. Mannitol
fermenters also produce a zone of yellow around mannitol + species. The Bacillus specie’s
reaction to the presence of lecithin and mannitol in the agar will help in its identification. The
presence of polymyxin B in the agar also helps to inhibit the growth of non-Bacillus organisms.
!
SHEEP BLOOD AGAR (SBA)
Universal plating medium for the isolation of bacteria including fastidious organisms.
Supports growth under aerobic and anaerobic conditions and demonstrates hemolysis. A base of
brain heart infusion supplemented with 5% sheep or 5% cow blood.
!
!
TINSDALE AGAR
Used in the isolation of Corynebacterium, especially C. diphtheriae. The tellurite inhibits
growth of most gram negative normal flora. C. diphtheriae can be differentiated from the
saprophytic corynebacteria by the production of a brown halo surrounding the grayish black
colonies. Other bacteria may grow on this media also, such as Staphylococcus, but will not have
the halo.
!
XYLOSE, LYSINE AND DEOXYCHOLATE (XLD)
Used for the isolation of Shigella spp. and Salmonella spp. from clinical samples.
Fermenters produce yellow colonies. Xylose fermenters that do not utilize the lactose or sucrose
produce amber colonies. Shigella, Provdencia and Proteus do not use any of the sugars, and
produce clear colonies. Proteus and Salmonella, which produces hydrogen sulfide, have black
centers.
!
! APPENDIX 4
! !
! TUBE MEDIA, BROTHS, AND AGAR SLANTS
!
BROTHS
!
Inoculation protocol:
Unless stated, all broths are inoculated with an isolated colony and incubated at 37oC for 24
hours; all slants are streaked ONLY and all agar plugs and stabbed ONLY.
!
BRAIN HEART INFUSION BROTH (BHI)
General all purposed growth medium. Used for logarithmic growth for testing pigment
production, or as a stock for further testing.
!
CARBOHYDRATE BROTHS
glucose, mannitol, trehalose, melibiose, L-arabinose, rhamnose, D-xylose, D-sorbitol, inositol,
maltose, dulcitol, lactose.
For determining the ability to utilization of carbohydrates (sugars). The acidic end
product by fermentation is detected by observing a shift in pH. The media changes from purple
to green/yellow, with any color change considered positive.
METHOD:
Emulsify a loop full of a pure culture in 500ul blood broth.
After incubation, add 3-5 drops of blood broth culture to carbohydrate broth
Requires growth before the test can be read. If there is no change in 24 hours, re-incubate
for an addition 24 hours before considering reaction negative.
!
CHRISTENSEN’S UREA AGAR
Tests for the presence of the enzyme urease that hydrolyzes urea, releasing ammonia.
This is detected by a changing the media to a pink/red. Bacteria that produce less urease, the
change is first seen in the slant because the alkaline reaction is augmented by the amines formed
from the oxidative decarboxylation of the amino acids in the air-exposed portion of the medium.
Positive: Pink
Negative: Yellow/no color change
!
LOEFFLER’S SERUM MEDIUM
Used as an isolation medium for clinical samples suspected of C. diphtheriae. The media
enhances granule formation seen with a methylene blue stain. Since colonies do not show any
differential characteristics, they are subcultured to tellurite based media for further identification.
It is also a general media to demonstrate protolytic activity of Actinomyces pyogenes and
Pseudomonas aeruginosa.
METHYL RED-VOGES PROSKAUER (MRVP)
Identifies one of two fermentation pathways of glucose: mixed acid or butanediol (or
butylene glycol) fermentations. Separates coliform bacteria and is used in testing feces
contaminated food and drinking water. Aids in the differentiation between Staphylococcus (VP+)
and Micrococcus (VP-) and Streptococcus species. Most Enterobacteriaceae utilize a mixed acid
pathway which is detected by the measuring the final acidity with the pH indicator, methyl red.
METHOD:
Inoculate broth with a pure culture and incubate for 18-24 hours.
Remove 2 ml
To the remaining culture, add the specified amount of methyl red
To the 2 ml, add the specified amount of a-naphthol (VP#1) and potassium
hydroxide solution (VP#2), respectively
Shake well for 30-60 seconds
Let stand for 10-15 minutes before confirming a negative reaction
Positive (MR): Red color (pH<4.4) Positive (VP): Rust colored color change
Negative (MR): Yellow color (4.4<pH<) Negative (VP): No color change
!
NITRATE BROTH
To test for the ability to reduce nitrates to nitrites or other reduction products by
extracting oxygen from nitrates.
METHOD
Inoculate broth with a loop full of pure culture and incubate for 24 hours.
Add 5 drops of sulfilic acid (Nitrate #1) and 5 drops of a- naphthylamine (Nitrate #2)
Positive: The red color develops within 30 seconds
Negative: No color change
NOTE: If no color reaction occurs, nitrates may not be reduced (true negative) or other end
products may be produced. To prevent false negatives, a small quantity of zinc is added. The
zinc will reduce the nitrates to nitrites. If the solution turns red, then it is true negative.
!
O/F MEDIA WITH GLUCOSE (OXIDATIVE-FERMENTATIVE)
Media to test for non-fermenters. The ratio of peptone to carbohydrates in addition to the
pH indicator of bromthymol blue, is different than in those media used to test fermenters.
METHOD
Two tubes are required for this test
After inoculation, one is overlaid with mineral oil while the second tube is left exposed to
air
INTERPRETATION
Fermentative bacteria produce acid on both tubes indicated by a change to a yellow media.
Oxidative bacteria can only grow in the open tube. Asaccharolytic organisms that do not utilize
carbohydrates produce no change in either tube.
ORTHONITROPHENYL-b-d-GALACTOPYRANOSIDE (ONPG)
Prompt identification of delayed lactose fermenters who may possess one of the two
enzymes tested: lactose permease and B-galactosidase. Permease is required to penetrate the
bacterial cell and the other cleaves the galactoside bond producing galactose and glucose. Non-
lactose fermenting bacteria do not have either enzyme.
METHOD: A loop full of pure culture is added
Positive: Intense yellow from the splitting of ONPG into d-galactose and yellow o-nitrophenol.
The reaction can be positive within 5-10 minutes to 60 minutes. Do not interpret as negative for
24 hours.
!
SELENITE F BROTH
An enrichment broth for the preferential growth of Salmonella spp. from feces. Lactose is
the main carbon source. Selenite inhibits growth of coliforms for up to 12 hours, allowing
Salmonella to replicate without competition. Ideally, subcultures will be made in the first 12-18
hours for maximum Salmonella recovery. The enrichment is void after 24 hours.
!
SIMMONS CITRATE AGAR
This medium contains sodium citrate which is the salt of citric acid, an organic
compound that is a metabolite in the Kreb’s cycle.
Growth without a change in color does not indicate a positive test result. For the bacteria
to be visible, they must be in log phase which is only possible if carbon and nitrogen is
assimilated. To confirm by color change, re-incubate for an additional 24 hours.
Positive: A deep blue color
Negative: No color change/green
!
SIM TEST MEDIUM
A semi-solid agar for the detection of motility, H2S and indole production in enteric
bacilli. H2S + organisms will produce H2S gas which reacts to the ferrous ammonium sulfate in
the agar to produce a ferrous sulfide, a black precipitate. After incubation, Kovak’s reagent is
added to test indole formation. Yersinia enterolitica, and Listeria monocytogenes require room
temperature incubation. Pseudomonas aeruginosa does not show the fanning out because it does
not grow well in the oxygen-deficient portions of the tube.
INTERPRETATION
Motility+/ H2S-: Growth at line of inoculation and throughout agar plug, no black ppt seen
Motility -/ H2S-: Growth at line of inoculation only, no black ppt seen
Motility +/ H2S+: Black ppt throughout agar plug
Motility - / H2S+: Thin black line at inoculation site
!
SODIUM CHLORIDE 6.5%
Various concentrations of salt can be used to differentiate species of Streptococcus,
Enterococcus or Mycobacterium. Sodium chloride added a nutrient broth. When the bacteria
utilize the glucose producing acid end products, the media will turn yellow, indicating growth
and salt tolerance.
Positive: Yellow
Negative: Purple
TRIPLE SUGAR AGAR IRON AGAR (TSI)
Used to differentiate fermentation pathways. Iron binds the hydrogen sulfide from the
sulfur containing precursors to form iron sulfide. By convention the letter “K” designates an
alkaline reaction and “A” stands for an acid reaction. “G” is for CO2 formation and H2S indicates
hydrogen sulfide production. The biochemical reactions (excluding gas and hydrogen sulfide
production) are outlined below. The TSI must be read within 18-24 hours. Longer incubation
times will result in fermentable carbohydrates becoming exhausted. The peptones and amino
acids will be utilized and alkaline substances will evolve. The small amount of acid produced by
glucose fermentation is oxidized rapidly in the slant which will remain or revert to alkaline.
In total, there are 6 possible reactions:
Note: The slant is read first, then the butt. A=acid; K=alkaline
1. A/AG+H- Glucose, lactose and/or sucrose utilized in slant, butt fermented with C02
2. K/AG- H+ Glucose only utilized in slant along with peptones; butt fermented with H2S.
There may be CO2
3. K/AG-H- Same as Rxn 2 without H2S
4. A/AG-H+ Glucose, lactose and/or sucrose utilized in slant butt fermented with H2S. There
may be CO2
5. K/KG-H- Glucose only utilized in slant along with peptones No butt fermentation.
6. A/AG-H- Glucose, lactose, and/or sucrose fermented without gas or H2S
There are 2 chambers in the tube. The SLANT portion is aerobic; the lower portion, the BUTT or
DEEP is protected from the air and relatively anaerobic.
!
Non fermenters
Without carbohydrate catabolism acids are not formed and the amine production in the
slant together with the alkaline buffers produces a “red” color throughout.
!
Glucose fermenters with the inability to ferment lactose
A small quantity of acid can be obtained form the low concentration of glucose in the
media. During the first 8-12 hours, this amount of acid may be sufficient to convert both the deep
and the slant to a yellow color. Within the next few hours the degradation of the amino acids
within the slant portion of the tube under the action of oxygen and bacteria begins to release
amines that soon counteract the small quantities of acid. In 18-24 hours the entire slant reverts to
an alkaline pH and returns to a red color. In the butt of the tube the amino acid degradation is
insufficient to counteract the acid formed and the medium remains yellow.
!
Non-sucrose fermenters.
The addition of sucrose in the media enables the screening of bacteria that do not utilize
lactose or sucrose. Here, the media will remain alkaline in the butt and slant portion of the tube.
!
Hydrogen sulfide production
Since this is colorless, the media contains sodium thiosulfate as an indicator. It is the
source of the sulfur atoms. The iron salts and the ferric ammonium citrate react with the H2S
production to produce an insoluble black precipitate (ferrous sulfide). An acid environment is
required to produce H2S which provides a source of H+ ions. Because the butt becomes acidic
with glucose fermentation (H+ ions increase) the black is seen there first. So the butt is always
read as “acid”.
APPENDIX 4
MEDIA RECIPES
From Difco and BBL manual
all recipes yield 1 L of media
Bile Esculin Agar Plates

Ingredient Amount Purpose
Beef extract 3.0 g Carbon, nitrogen, vitamin and mineral source
Enzymatic digest of
gelatin 5.0 g Carbon and ntirogen source.
Enterococci can hydrolyze esculin to esculetin
Esculin 1.0 g and dextrose. Streptococci cannot.
Oxgall inhibots the growth of other Gram-
Oxgall (bile) 20.0 g positive bacteria.
The esculetin reacts with ferric citrate to form a
Ferric Ammonium citrate 0.5 g dark brown/black complex.
Agar 14.0 g Solidifying agent.
Cornmeal agar with 1% tween 80 (CMA)

Provides nutrients to support the growth of
Corn meal infusion 2.0 g fungal species.
Stimulates the production of pseudohyphae in
Tween 80 1% Candida.
Hektoen Agar (HE)

Proteose Peptone 12.0 g Provides nitrogen source.
Provide nitrogen, carbon, amino acids and
Yeast extract 3.0 g vitamins.
Inhibit gram-positive organisms but also toxic to
Bile salts No. 3 9.0 g some gram-negative strains
1 of 3 fermentable carbon sources. Can allow for
Lactose 12.0 g differentiation of enterics.
Saccharose (sucrose) 12.0 g differentiation of enterics.
Salicin 2.0 g differentiation of enterics.
Sodium Chloride 5.0 g Maintain osmotic balance.
Sodium thiosulfate 5.0 g Can be reduced to hydrogen sulfide.
Reacts with hydrogen sulfide to form a black
Ferric ammonium citrate 1.5 g precipitate.
Bromthymol blue 65.0 mg pH indicator.
Acid Fuchsin 0.1 g pH indicator.
LB Agar, Miller
Tryptone 10.0 g Provides nitrogen source.
Sodium chloride 10.0 g Osmotic balance.
Loeffler's Medium
Proteose peptone 1.5 g Source of amino acids, nitrogen and peptides.
Sodium chloride 1.25 g Source of essential ions.
Dextrose 1.25 g Fermentable carbohydrate.
Beef Extract 0.75 g Source of amino acids, nitrogen and peptides.
Coagulates. This helps determine proteolytic
Horse Serum 750 mL activity of organisms.
Demineralized water 250 mL Fill to final volume of 1L.
MacConkey Agar
Casein 17.0 g Provides nitrogen source.
Peptone 3.0 g Provides nitrogen source.
Fermentable carbon source. Allows for the
differentiation of lactose fermenters from
Lactose 10.0 g nonlactose fermenters
Bile salts 1.5 g Inhibits the growth of Gram-positive organisms.
Neutral red 0.03 g pH indicator.
Crystal violet 1.0 mg Inhibits the growth of Gram-positive organisms.
Mannitol Salt Agar
(MSA)
Casein 5.0 g Provides nitrogen source.
Peptic digest 5.0 g Provides nitrogen source.
Partial or complete inhibition of non-
Sodium chloride 75.0 g staphylococcal species.
Fermentable carbon source. Allows
D-mannitol 10.0 g differentiation of Staphylococcal species.
Phenol Red 25.0 mg pH indicator.
Mannitol Yolk Polymixin B (MYP)

Fermentable carbon source. Allows for
D-mannitol 10.0 g differentiation of B. cereus and B. subtilis.
Phenol Red 25.0 mg pH indicator.
Source of lecithin. Allows for differentiation of
Egg Yolk Enrichment 12.5 mL B. cereus and B. subtilis.
250,000 Antimicrobial, inhibits the growth of most other
Polymixin B units bacteria.
Mueller-Hinton Agar (M-H)

Acid digest of casein 17.5 g Provides nitrogen source.
Acts as a protective colloid against toxic
Starch 1.5 g sustances that may be present.
Nutrient Agar (NA)

Potato Flake Agar
(PFA)
Potato Flakes 20.0 g Nutritive base.
Dextrose (glucose) 10.0 g Carbon and energy source.
Pseudomonas Agar P (Tech Agar, P agar)

Pancreatic digest of
gelatin 20.0 g Provides nitrogen and amino acid source.
Magnesium chloride 1.4 g Promote pyocyanin production.
Potassium sulfate 10.0 g Promote pyocyanin production.
Glycerol 10.0 g Energy source. Enhances pigment production.
Regan-Lowe Charcoal
Agar
casein 10.0 g Provides nitrogen source.
Neutralize toxic substances such as fatty acids
Soluble starch 10.0 g and peroxides.
Sodium chlroide 5.0 g Osmotic balance.
Neutralize toxic substances such as fatty acids
Charcoal 4.0 g and peroxides.
Vitamin that supports growth of Bordetella
Niacin 0.01 g species.
Proviedes nutrients to support the growth of
Defibrinated horse blood 10% Bordetella species.
Sheep's Blood agar, 5% (SBA)

Heart muscle 2.0 g Provides nitrogen, amino acids and vitamins.
Provides nitrogen, carbon, amino acids and
Yeast Extract 5.0 g vitamins.
Provides additional growth factors for fastidious
organisms. Allows differentiation of bacteria
Defibrinated Blood 5% based on their ability to hemolyze RBCs.
SIM agar
Pancreatic digest of Provides nitrogen source. Rich in tryptophan,
casein 20.0 g which can be used to produce indole.
Peptic digest 6.1 g Provides nitrogen source.
Ferrous ammonium
sulfate 0.2 g Indicators of hydrogen sulfide production.
Sodium thiosulfate 0.2 g Indicators of hydrogen sulfide production.
Low agar concentrations allow for motility
Agar 3.5 g through the semisolid medium.
Simmons Citrate Agar

Ammonium dihydrogen
phosphate 1.0 g Sole source of nitrogen.
Dipotassium phosphate 1.0 g Buffer.
Sodium chloride 5.0 g Maintain osmotic balance.
Sodium citrate 2.0 g Sole source of carbon.
Magnesium sulfate 0.2 g
Bromthymol blue 0.08 g pH indicator.
Tinsdale Tellurite Agar

Provides nitrogen, vitamins, carbon and amino
Proteose peptone No. 3 20.0 g acids.
Sodium chloride 5.0 g Maintain osmotic balance.
Bovine Serum 69.86 mL Provides essential growth factors.
Horse Serum 79.72 mL Provides essential growth factors.
L-Cystine 0.42 g Provide sulfur for hydrogen sulfide production.
Sodium Thiosulfate 0.59 g Provide sulfur for hydrogen sulfide production.
Selective agent. Also reacts with hydrogen
Potassium Tellurite 0.29 g sulfide to form a brown halo of metallic tellurite.
Triple Sugar Iron Agar (TSI)

Provides nitrogen, vitamins, carbon and amino
Proteose Peptone No. 3 5.0 g acids.
1 of 3 fermentable carbon sources. Note that this
Dextrose (glucose) 1.0 g one is lower.
Lactose 10.0 g 1 of 3 fermentable carbon sources.
Sucrose 10.0 g 1 of 3 fermentable carbon sources.
Ferrous sulfate 0.2 g Allows for the detection of hydrogen sulfide.
Sodium thiosulfate 0.3 g Provide sulfur for hydrogen sulfide production.
Phenol red 24.0 mg pH indicator.
Urea Agar
gelatin 1.0 g Source of nitrogen, carbon and amino acids.
Dextrose 1.0 g Energy source.
Potassium phosphate 2.0 g Buffer.
Urea 20.0 g Nitrogen source if urease-positive.
Phenol red 12.0 mg pH indicator.
XLD
Yeast extract 3g Source of nitrogen, carbon and vitamins.
1/3 fermentable carbohydrates. Salmonella
Lactose 7.5 g cannot ferment.
1/3 fermentable carbohydrates. Salmonella
Sucrose 7.5 g cannot ferment.
Xylose 3.5 g 1/3 fermentable carbohydrates. Most enterics,
including Salmonella can ferment.
Salmonella can decarboxylate lysine. This allows
L-lysine 5g for a reversion to alkaline conditions.
Allows for detection of hydrogen sulfide under
Ferric ammonium citrate 0.8 g alkaline conditions.
Phenol red 0.08 g pH indicator.
Sodium chloride 5g Osmotic balance.
Sodium deoxycholate 2.5 g Selective agent.
Sodium thiosulfate 6.8 g Can be reduced to hydrogen sulfide.
APPENDIX 6
!
ANTIBIOTIC DISC DIFFUSION TEST
ANTIMICROBIAL SENSITIVITY TESTING
An organism’s sensitivity or resistance to a given antimicrobial agent is determined with
the Kirby-Bauer test. The size of the zone of sensitivity is meaningless without a comparison to
references derived from a correlation between the numbers of bacteria killed and the
concentration of the drug used. The reference values are determined by testing many strains
against a particular antibiotic and then establishing a regression line. These strains were chosen
because they have susceptibility end points in the mid range of the concentrations tested and
have minimal tendencies to change susceptibility over time. The minimum inhibitory
concentration (MIC) is determined for any zone size. Often, a zone may be established where the
MIC cannot be determined. The organism is then said to be intermediate to susceptibility, with
less than 5% falling into this range. The MIC should be reached at the site of infection (without
toxicity to the host) usually attempting to reach levels 4-8 times greater that MIC if possible.
!
!
!
ANTIMICROBIAL SENSITIVITY- DISC DIFFUSION ASSAY
This test determines the lowest concentration of an antibiotic that inhibits growth of the
organism. Interpretations are derived from comparisons with reference strains. The importance
of this test is:
(1) The criteria that the bacteria must be able to grow well after overnight incubation in air.
(2) Reproducibility between laboratories and
(3) As a guide to the physician in selecting the proper antibiotic and dose for a given infection
including patient history, sight of infection and mode of delivery.
!
METHOD
Four or five isolated colonies are grown in broth to log phase or emulsified in normal
saline to a concentration of 15x108 CFU/ml, or a comparative turbidity to 0.5 MacFarland
standard (Appendix 2). A sterile cotton swab is dipped in the cell suspension then rolled against
the inside of the tube to eliminate excess liquid. The swab is streaked in 4 different directions
(refer to plating methods) to form a lawn of growth on a Mueller-Hinton plate and allowed to
dry. Several types of antibiotic discs are spaced around the plate using a disc dispenser. The plate
is then incubated for 24 hours.
!
INTERPRETATION
Growth is inspected. It is necessary to have even and confluent growth over the entire
plate before interpretation. Each zone of clearing is measured and compared to the known MIC
interpretive standards.
Susceptible: The organism should respond to the usual administered dose
Moderately susceptible: The organism may be inhibited by the maximal dose given, but this
would not be the first drug of choice
Intermediate: The information obtained is inconclusive or equivocal
Resistant: The organism is not inhibited by any acceptable concentrations
Difficulties may arise in regards to interpreting the results due to a poorly prepared plate.
1. If there are unevenly spaced discs, the zones will overlap and interpretation of the exact size
will be questionable. Oval or elliptical zones may result with difficulty in determining whether to
measure the long or the short diameters.
2. If the streaking is poor with space between the streaks, and no confluent growth, the margins
will not be distinct and the measurement will be inexact.
3. There may be colonies growing within the zone. It must be determined if these are a resistant
mutant strain or a contaminating organism from a mixed culture requiring additional biochemical
tests on these colonies.
!
Antimicrobial Agents Table
! ! ! !
! b-cidal vs
! lipid actively enzymatic
!
action pH sensitivity
b-static soluable secreted inactivated
! ! ! ! ! !
penicillins cidal cell wall ! kidney penicillinase range of pH
! ! ! !
vancomycin cidal cell wall ! kidney ! !
! ! ! ! !
cephalosporins cidal cell wall
! ! cephalosporinases range of pH
! !
! ! metabolic
! ! decrease affinity of !
trimethoprim cidal + kidney
analogs target
! ! !
protein ! ! ! !
gentamycin static kidney ineffecient transport
synthesis
! ! !
protein ! !
active
! !
erthryomycin static alteration in the 50S neutral pH
synthesis efflux
! ! !
! !
! !
protein liver
chloramphenicol static acetyltransferase
synthesis excretion
! ! !
protein
! !
active
!
decreas affinity to
!
acidic
tetracyclines static
synthesis efflux 30S pH
! ! ! !
oxicillin cidal cell wall ! ! b-lactamase !
! ! ! ! ! !
!
! ! protein
! phosphorilation;
kanamycin cidal kidney acetylation;
synthesis
adenylation
! ! ! ! ! !
!
! ! protein
! phosphorilation;
streptomycin static kidney acetylation;
synthesis
adenylation
! ! !
metabolic
! ! ! !
triple sulfa static
analogs
APPENDIX 7
!
!
LANCEFIELD GROUPING FOR STREPTOCOCCUS
!
The grouping system is based upon detecting of extracted soluble antigens. They are either cell
wall polysaccharides as in group A, B, C, F and G or cell wall lipoteichoic acids in group D and
Enterococcus species. There are group specific and type specific in the b-hemolytic
Streptococcus. The S.viridans and non-hemolytic are too diverse and are not considered part of
the Lancefield groupings. The group specific antigen (C substance) is polysaccharide and an
integral part of the bacterial cell.
!
GROUP SPECIES HEMOLYSIS HABITAT SEQUELAE ID TESTS
!
A S. pyogenes β pharynx;skin acute pharyngitis Bacitracin A
wound cellulitis Agglutination
impetigo
septicemia
rheumatic fever
glomerulonephritis
rheumatic
endocarditis
B S. agalactiae β (or γ) pharynx; vagina puerperal sepsis Hippurate hydrolysis

! ! ! stool endocarditis Bile-esculin
! ! ! newborn-several pneumonitis CAMP
! ! ! ! neonatal infection !
! ! ! ! pneumonia !
! ! ! ! meningitis !
! ! ! ! septicemia !
C S. equi β pharynx; vagina wound Hippurate hydrolysis

! S. equisimilis β skin perperal sepsis Glycerol ferm
! S. sysgalactiae α ! cellulitis Trehalose ferm
! ! ! ! endocarditis Sorbitol ferm
D Enterococcus γ large bowel UTI NaCl +

! E. faecalis (α) ! pelvis abscesses Bile Esculin
! E. faecium (β) ! peritonitis !
! E. durans ! ! wound infections !
F Non-enterococcus endocarditis
S. bovis α, γ
S. equinus (α)
S. anginosis β mouth; teeth sinusitis
pharynx dental caries
meningitis
brain abscesses
pneumonia
!
G S. canis β pharynx; vagina puerperal infection Arginine -NH3
skin wound infection Inulin
! ! ! ! ferm !
! ! ! ! endocarditis !
H S. sanguis α mouth; teeth dental caries polysac. in 5%

! pharynx endocarditis sucrose broth
! brain abscesses !
! septicemia !
K S. salivarius α pharynx; mouth endocarditis Glucose,sucrose

septicemia maltose-acid +
sinusitis Glycerol,
mannitol,
meningitis sorbitol-acid-
!
None S. pneumonia α pharynx; mouth lobar pneumonia Optichin -S
trachea septicemia
Quellung !
otitis media Bile solubility
meningitis
endocarditis
S. viridans α Optichin -R
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(Please!list!procedures!in!the!order!in!which! (Please!include!details!about!the!results.!For!
they!were!performed.!If!asked!to!make!a! example,!“bubbles!formed!upon!the!
flow!chart,!please!attach!the!flow!chart!to! addition!of!H2O2!=!catalase!+”!instead!of!
this!sheet.)! just!saying!“catalase!+”.)!
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!
Material(for(this(manual(was(compiled(from:(
!
Benathen,!I.!A.,!Microbiology!with!Health!Care!Applications!Belmont,!Ca,!1993!Star!
!
Brock,!T.!D.,!Madigan,!M.T.,!Martinko,!J.M.,!Parker,!J.,!Biology!of!Microorganisams,!ed.!7!New!
Jersey,!1994!PrenticeIHall!
!
Edinburgh!University,!Microbiology!3BH!Lab.!ExercisesIIsolation!of!Food!Poisoning!Organisms!
Edinburgh,!U.K.,!1979,!Edinburgh!University!
!
Goodman!G.A.,!Goodman,L.S.,!Gilman,A.,!The!Pharmacological!Basis!of!Therapeutics,!ed.!6!New!
York,!NY,!1980,!Macmillan!
!
Hirsh,!D.C.,!Carlson,!J.,!Snipes,!K.,!Beaman,!B.,!Miller,!C.H.,!Pappagianis,!D.,!LeFebvre,!R.!
Lecture!Syllabus,!Veterinary!Microbiology!127!U.C.!Davis!School!of!Veterinary!
Medicine,!Davis,!Ca.!1994!
!
Jang,!S.!S.,!Biberstein,!E.!L.,!Hirsh,!D.C.,!A!Diagnostic!Manual!of!Veterinary!Clinical!Bacteriology!
and!Mycology!U.C.!Davis!School!of!Veterinary!Medicine,!Davis,!Ca.!1994!
!
Jawetz,!E.,!Melnick,!J.L.,!Adelberg,!E.A.,!Review!of!Medical!Microbiology,!ed.!7!Los!Alato,!Ca,!
1987!Appelton!and!Lange!
!
Koneman,!E.W.,!Allen,!S.!D.,!Janda,!W.!M.,!Schreckenberger,!P.!C.!Winn,!W.!C.,!Color!Atlas!and!
Textbook!of!Diagnostic!Microbiology,!ed.!5!Philadelphia,!Penn.,!1997,!Lipponcott!
!
Leboffe,!M.!J.,!Pierce!B.E.!A!Photgraphic!Atlas!for!the!Microbiology!Laboratory,!ed.!4!2011!
Morton!Publishing!Company.!
!
Murry,!M.!R.,!Rosenthal,!K.!S.,!Kobayashi,!G.!S.,!Pfaller,!M.A.,!Medical!Microbiology!ed.!3!St!
Louis,!Mo.!1998,!Mosby!
!
Prescott,!L.!M.,!Harley,!J.P.,!Klein,!D.A.,!Microbiology,!ed.!2.!Dubuque,!Ia,!1993,!William!C.!
Brown!
!
Roitt,!I.,!Brostoff,!J.,!Male,!D.,!Immunology,!Second!Edition!New!York,!N.Y.,!1989,!Gower!
Medical!Publishing!
!
Salyers,!A.A.,!Whitt,!D.D.,!Bacterial!Pathogenesis:!A!Molecular!Approach!Washington!D.C.,!1994,!
ASM!Press!
!
Schaechter,!M.,!Medoff,!G.,!Schlessinger,!D.,!!Mechanisms!of!Microbial!Disease!Baltimore,!Md.,!
1989,!Williams!and!Wilkins!
!
Varnam,!A.H.,!Evans,!M.G.,!Foodborne!Pathogens;!An!Illustrated!Text!St.!Louis,!Mo.,!1991,!
Mosby.!
!
Warinner,!P.!Q.,!Clinical!Microbiology!Review!Long!Island,!N.!Y.,!1995,!Wysteria,!Ltd!
!
Winter,!L.,!Laboratory!Exercises!in!Pathogenic!Bacteriology!Ithaca,!N.Y.,!1985,!New!York!State!
College!of!Veterinary!Medicine,!Cornell!University!
!
Youmans,!G.!P.,!Paterson,!P.Y.,!Sommers,!H.!M.,!The!Biological!and!Clinical!Basis!of!Infectious!
Diseases!ed.!3!Philadelphia,!Penn.,!1985,!Saunders!
!
!
!
!
!
!
Useful!summaries!found!in!Medical!Microbiology!
!
Table!21I1! page!170I71! Specimen!Collection!for!Bacterial!Pathogens!
Table!45I1! page!372I75! Summary!of!Bacteria!Associated!with!Human!Disease!
Table!45I2! page!375! Selected!Bacteria!Associated!with!Fooborne!Disease!
Table!45I3! page!375! Selected!Bacteria!Associated!with!Waterborne!Disease!
Table!45I4! page!376! Arthropod!Associated!Disease!
!

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MISCELLANEOUS TESTS AND REAGENTS

Bile Esculin Agar Plates

Cornmeal agar with 1% tween 80 (CMA)

Hektoen Agar (HE)

Mannitol Yolk Polymixin B (MYP)

Mueller-Hinton Agar (M-H)

Nutrient Agar (NA)

Pseudomonas Agar P (Tech Agar, P agar)

Sheep's Blood agar, 5% (SBA)

Simmons Citrate Agar

Tinsdale Tellurite Agar

Triple Sugar Iron Agar (TSI)

B S. agalactiae β (or γ) pharynx; vagina puerperal sepsis Hippurate hydrolysis

C S. equi β pharynx; vagina wound Hippurate hydrolysis

D Enterococcus γ large bowel UTI NaCl +

H S. sanguis α mouth; teeth dental caries polysac. in 5%

K S. salivarius α pharynx; mouth endocarditis Glucose,sucrose

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