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(Practical Manual)

MIC 101

Prepared by: M.Homam.Jarkash (Microbiologist )

Supervised by : Dr.Sameh Rabia(Pharmacist )

I hope this brief handout in practical pharmaceutical microbiology

to be beneficial and will informative for pharmacy diploma in the health
It concerns about general aspects in pharmaceutical microbiology,
for example: sterilization and disinfection, Gram staining and
microscopy, general microbiological procedures, medical specimens
collections and preparation of different important media.
I am asking Allah for conciliation to all of us.

General safety

Some of the microorganisms used in this course may be pathogenic

for humans and animals. As a result, certain rules are necessary to avoid
the possibility of infecting yourself or other people.
A. Microbiological procedures:
1. reporting all spills and broken glassware to the instructor and
receiving instructions for cleanup
2. methods for aseptic transfer
3. minimizing or containing the production of aerosols
4. washing hands prior to and following laboratories and at any
time contamination
5. never eating or drinking in the laboratory
6. disinfecting lab benches prior to and at the end of lab session
7. identification and proper disposal of different types of waste
8. never applying cosmetics, including contact lenses, or placing
objects (fingers, pencils) in the mouth or touching the face
9. good lab practice, including returning materials to proper
locations, proper care and handling of equipment.

B. Protective procedures:
1. tying long hair back, wearing personal protective equipment (eye
protection, coats, closed shoes; glasses may be preferred to contact
2. always using appropriate pipetting devices and understanding
that mouth pipetting is forbidden
C. Emergency procedures:
1. locating and properly using emergency equipment (eye-wash
stations, first-aid kits, fire extinguishers, chemical safety showers,
telephones, and emergency numbers)
2. reporting all injuries immediately to the instructor.

Universal Precautions and Laboratory Safety Precautions:

1- All specimens of blood and body fluids should be put in a well-
constructed container with a secure lid to prevent leaking during

2- All persons processing blood and body-fluid specimens should
wear gloves.
3- For routine procedures, such as histologic and pathologic studies
or microbiologic culturing, a biological safety cabinet is not necessary.
4- Mechanical pipetting devices should be used for manipulating all
liquids in the laboratory.
5- Use of needles and syringes should be limited to situations in
which there is no alternative.
6- Laboratory work surfaces should be decontaminated with an
appropriate chemical germicide after a spill of blood or other body
fluids and when work activities are completed.
7- Contaminated materials used in laboratory tests should be
decontaminated before reprocessing or be placed in bags and disposed
of in accordance with institutional policies for disposal of infective
8- Scientific equipment that has been contaminated with blood or
other body fluids should be decontaminated and cleaned before being
repaired in the laboratory or transported to the manufacturer.
9. All persons should wash their hands after completing laboratory
activities and should remove protective clothing before leaving the
10. There should be no eating, drinking, or smoking in
the work area.

Session 1:
Sterilization and Disinfection:

Sterilization: is the killing of all microorganisms in a material or on the

surface of an object
- A surface or an object is either sterile or it is not sterile, there are no
gradations in sterility
Disinfection: means reducing the number of viable microorganisms
present in a sample
- Not all disinfectants are capable of sterilizing, but, of course, all
disinfectants are employed with the hope of disinfecting.
Sanitization: is the cleaning of pathogenic microorganisms from public eating
utensils and objects such as that done by the kitchen of a restaurant.
Disinfectant: is a chemical or physical agent that is applied to inanimate
objects to kill microbes.
Antiseptic: is a chemical agent that is applied to living tissue to kill microbes
- Note that not all disinfectants are antiseptics because an antiseptic
additionally must not be so harsh that it damages living tissue.
- Examples of Specific antimicrobial agents that used as
disinfectant or antiseptic:
(i) Surfactants: soaps
(ii) Various organic acids and bases: benzoic acid
(iii) Heavy metals: mercury (mercurochrome)
(iv) Halogen-containing compounds: Iodine, Chlorine
(v) Alcohols: ethyl alcohol, propyl alcohol
(vi) Phenol and phenol derivatives: Phenol, Dettol ®
(vii) Oxidizing agents: Hydrogen peroxide
(viii) Alkylating agents: formaldehyde, glutaraldehyde.
(ix) Certain dyes: Gentian violet.
-Methods of Sterilization:
1- Heat:
Heat is a highly efficient means of sterilization so long as the material
to be sterilized is resistant to heat.
- Different types of heat application include:
A- Dry heat:

To effect sterilization dry heat typically requires higher temperatures
than moist heat It also is less penetrating and requires longer exposure.
-Typically one bakes materials in an oven at
(i) 171C for at least one hour
(ii) 160C for at least two hours
(iii) 121C for at least 16 hours
Incineration is another common method of dry heat sterilization, e.g.,
such as the flame incineration of an inoculating loop.

Figure 1: Different types of dry heat ovens.

B- Moist heat
Moist heat is more effective than dry heat at a given temperature or
length of exposure Moist heat is also more penetrating than dry heat.
However, to achieve sterilization employing moist heat requires
rather elaborate equipment, i.e., the employment of an autoclave.
- Autoclave:
is a high pressure device used to allow the application of moist heat
above the normal-atmosphere boiling point of water. Exposure to 121C
for 15+ minutes is typically sufficient to sterilize, the material must be

121C before the clock starts, Large items, large volumes, and items
that are poorly penetrated by steam may take much longer than 15
minutes to sterilize.

Figure 2: Different types of autoclaves.

C- Pasteurization:
is the application of moist heat of less-than boiling temperatures to
foods to prevent the growth of food-spoiling organisms as well as
various heat-labile pathogens.

2- Ultraviolet (UV) radiation:

UV light is not terribly penetrating but is good for disinfecting surfaces
and air.
3- Ionizing radiation
Ionizing radiation is radiation that ionizes water; this temporarily turns
water into an oxidizing agent.
4- Strong visible light
Strong visible light can negatively affect bacterial viability so excessive
exposure to strong visible light should be avoided when one's goal is
culture preservation
5- Filtration (HEPA filters)
Filtration is a common means of antimicrobial treatment used when
materials are heat labile. High-efficiency particulate air (HEPA) filters
are used to filter the air flowing into aseptic environments and out of
potentially contaminated ones (e.g., containment facilities).
6- Gas Sterilization:
It is a common means of antimicrobial treatment used when materials
are heat and filtration labile. The most common used gas is: Ethylene

Effect of disinfectant –Soap- on Bacteria (Hand


Learning Objectives:
1. Understand the value of proper hand washing
2. Understand the importance of efficient decontamination procedures
3. Evaluate the effectiveness of hand washing and decontamination
4. Perform routine microbiological monitoring.
Most commercial hand washing products contain antibacterial
chemicals. Proper hand-washing technique performed by clinical
personnel is the most effective method of controlling infections,
especially nosocomial infections. A layer of oil and the structure of the
skin prevent the removal of microorganisms by simple hand washing.
Using a soap or gel will help remove the oil, and scrubbing with a brush
for 7 to 8 minutes will maximize the removal of both
transient(contaminated) and resident microorganisms.
1st Period:
1- Before doing any hand washing, using one of your hands, gently
make a five-finger impression on one of the TSA plates by rolling each
finger and your thumb on the agar. Label this plate with your name,
date, and “before”.
2- Take one of the soaps supplied and wash your hands according
to the directions of your instructor. Wash your hands for the length of
time assigned by the instructor. Some appropriate intervals are 10
seconds, 30 seconds, and 3 minutes.

3- After washing, make another five-finger impression (using the
same hand and fingers) on a different TSA plate. Mark this plate with
your name, date, and the duration of hand washing and label it "after".
4- Incubate both plates at 35°C for 24 to 48 hours.
2 Period:
Examine the TSA plates around the area where the finger
impressions were made. Compare the “before” and “after” plates.
a. Type of hand-washing material used:
b. Length of time of hand washing:
c. Type of culture medium used:
d. Hours of incubation:
e. Temperature of incubation:
f. Colony count before to hand washing:
g. Colony count after hand washing:
h. Colony shape :
I. Interpretation:

Session 2:
Microscopy and bacterial staining

The microscope
The microscope is a devise that magnifies the small unseen
objects. So in order to study microbes we have to use microscopes.
Types of microscopes:
1- light microscopes: 2 types
2- electron microscopes(EM): 2 types
The light microscope
The light microscope uses the light and lenses to illustrate and
magnified the objects. The simple light microscope consists of one lens
that used to magnify the objects. The compound light microscope uses
two systems of lenses.
The typical objectives have powers of 10X, 40X, and100X.
Figure1.1: the compound microscope

In the compound microscope light

from the source of light (lamp) is
collected by condenser lens and
pass to the specimen. The light
pass the specimen to the objective
lens that magnifies the image and
passes it to the ocular lens that
magnifies the image again and
passes it to the eye. So the
magnification power of a
microscope is obtained by
multiplying the magnification
power of objective lens by the
magnification power of eye lens.
The typical ocular lens has
magnification power of 10X.

Electron microscope
The EM was firstly made in Germany (1930-1933); but it began
to be used for the studying of cells and tissue at 1952.The EM uses the
electron beam instead of light.
Microscopic measurement of organisms:
It frequently is necessary to accurately measure
the size of the microorganism one is viewing. The
size of microorganisms is generally expressed in
metric units and is determined by the use of a
microscope equipped with an ocular micrometer.
An ocular micrometer is a small glass disk on
which uniformly spaced lines of unknown distance,
ranging from 0 to 100, are etched. The ocular
micrometer is inserted into the ocular of the
microscope and then calibrated against a stage
micrometer, which has uniformly spaced lines of
known distance etched on it. The stage micrometer
is usually divided into 0.01 millimeter and 0.1
millimeter graduations. The ocular micrometer is
calibrated using the stage micrometer by aligning
the images at the left edge of the scales.
The dimensions of microorganisms in dried,
fixed, or stained smears tend to be reduced as much
as10 to 20% from the dimensions of the living
microorganisms. Consequently, if the actual
dimensions of a microorganism are required,
measurements should be made in a wet-mount.

Applications of light microscope

1- Wet mount preparation: in this process a drop of the microbial
suspension is placed on slide and covered by coverslip then it will
examined by the light microscope.
2- Hanging-drop: a drop of the microbial suspension is placed on the
coverslip, which is then inverted over the dip in a depression
slide. Then it will be examined by the light microscope. The two

Review questions
Procedures that are needed to see microorganism by light microscope

Draw a representative field for each bacterium.



Use Magnification: X10 then Magnification: X40and X100

Session 3:
The bacterium cell is very small it range from 0.1µm to 10 mm but
there are some large bacteria that can be as long as 100µm.In order to
study the size and shape of bacteria the bacteria fixed on a slide then it
must be stained to get a contrast between the bacteria and its
surrounding medium.
Simple stain
Bacteria can be stained using basic dyes like crystal violet,
carbolfuchsin, and methylene blue. Simple dyes used for staining the
whole cell or a part of it: example the spores can be stained by
malachite green or carbolfuchsin. Crystal violet and others stain the
3whole cell.

Differential stains
Use to distinguish between two or more cell types. Gram stain and
acid-fast stain are other deferential stains
Gram stain
The cell wall is stained in this process. Bacteria are stain as
1- stain with basic dye
2- iodine is added as a mordant for the stain
3- rinse with alcohol
4- stain with counter stain of deferent color of the initial stain.
If the bacteria appear with color on initial stain it said to be gram-
positive, and if appear with color of the counter stain it said to be gram-
negative bacteria.
This is depends on the amount of peptidoglycan in the cell wall. gram-
positive bacteria have large amount of peptidoglycan so it will absorbs
the initial stain and alcohol can rinse it. Gram-negative bacteria have
small amount of peptidoglycan so it will absorbs small amount of the
initial stain so alcohol rinse it and it will absorbs the counter stain and
retained its color.

Acid-fast stain
It used in the identification of mycobacterium species.
Mycobacterium is stained as follow:
1- stained with hot carbolfuchsin (red dye)
2- washed with acidified alcohol
3- stained with methylene blue
Acid-fast bacteria contain a wax-like material that binds a primary
dye such as carbolfuchsin, even when they washed by acidified alcohol.
So it will appear with red color. Nonacid- fast bacteria initially stained
with the red dye but are decolorized by alcohol then it will stain with
methylene blue. So it will appear with blue color.

Review questions

1.What is the difference between a simple and differential stain?

2. Name the reagent used and state the purpose of each of the following in
the Gram stain:
a. mordant
b. primary stain
c. decolorizer
d. counterstain
3. Which step is the most crucial or most likely to cause poor results in the
Gram stain? Why?


4. Why must young cultures be used when doing a Gram stain?

5.What is the purpose of the heat during the acid-fast staining procedure?

6. What is the function of the counterstain in the acid-fast staining


Make a drawing of the distribution of the colonies

Session 4:
Growth medium

A growth medium or culture medium is a liquid or gel designed to

support the growth of microorganisms or cells [1], or small plantslike
the moss Physcomitrella patens [2]. There are different types of media
for growing different types of cells.
There are two major types of growth media: those used for cell culture,
which use specific cell types derived from plants or animals,
andmicrobiological culture, which are used for growing
microorganisms, such as bacteria or yeast. The most common growth
media for microorganisms are nutrient broths and agar plates;
specialized media are sometimes required for microorganism and cell
culture growth.[1] Some organisms, termed fastidious organisms,
require specialized environments due to complex nutritional
requirements. Viruses, for example, are obligate
intracellular parasites and require a growth medium containing living
Types of growth media
The most common growth media for microorganisms are nutrient
broths (liquid nutrient medium) or LB medium (Lysogeny Broth).
Liquid media are often mixed with agar and poured intopetri dishes to
solidify. These agar plates provide a solid medium on which microbes
may be cultured. They remain solid, as very few bacteria are able to
decompose agar. Bacteria grown in liquid cultures often
form colloidal suspensions.
A. Minimal media
Minimal media are those that contain the minimum nutrients possible
for colony growth, generally without the presence of amino acids, and
are often used by microbiologists and geneticists to grow "wild type"
microorganisms. Minimal media can also be used to select for or
againstrecombinants or exconjugants.
Minimal medium typically contains:

a carbon source for bacterial growth, which may be a sugar such

as glucose, or a less energy-rich source like succinate
various salts, which may vary among bacteria species and
growing conditions; these generally provide essential elements such
as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria
to synthesize protein and nucleic acid
B. Selective media

Selective media are used for the growth of only select microorganisms.
For example, if a microorganism is resistant to a certainantibiotic, such
as ampicillin or tetracycline, then that antibiotic can be added to the
medium in order to prevent other cells, which do not possess the
resistance, from growing. Media lacking an amino acid such
as proline in conjunction with E. coli unable to synthesize it were
commonly used by geneticists before the emergence
of genomics to map bacterial chromosomes.
Selective growth media are also used in cell culture to ensure the
survival or proliferation of cells with certain properties, such
as antibiotic resistance or the ability to synthesize a certain metabolite.
Normally, the presence of a specific gene or an allele of a gene confers
upon the cell the ability to grow in the selective medium. In such cases,
the gene is termed a marker.

Some examples of selective media include:
Eosin-methylene blue agar (EMB) that contains methylene blue –
toxic to Gram-positive bacteria, allowing only the growth of Gram
negative bacteria
YM (yeast and mold) which has a low pH, deterring bacterial
Blood agar (used in strep tests), which contains bovine heart
blood that becomes transparent in the presence of
MacConkey agar for Gram-negative bacteria
Hektoen enteric agar (HE) which is selective for Gram-negative
Mannitol salt agar (MSA) which is selective for Gram-positive
bacteria and differential for mannitol
Terrific Broth (TB) is used with glycerol in cultivating
recombinant strains of Escherichia coli.
Xylose lysine desoxyscholate (XLD), which is selective for
Gram-negative bacteria
Buffered charcoal yeast extract agar, which is selective for
certain gram-negative bacteria, especially Legionella pneumophila
C. Differential media
Differential media or indicator media distinguish one microorganism
type from another growing on the same media.[5] This type of media
uses the biochemical characteristics of a microorganism growing in the
presence of specific nutrients or indicators (such as neutral red, phenol
red, eosin y, or methylene blue) added to the medium to visibly indicate
the defining characteristics of a microorganism. This type of media is
used for the detection of microorganisms and by molecular biologists to
detect recombinant strains of bacteria.
Examples of differential media include:
Eosin methylene blue (EMB), which is differential for lactose
and sucrose fermentation
MacConkey (MCK), which is differential for lactose
mannitol salt agar (MSA), which is differential for mannitol
X-gal plates, which are differential for lac operon mutants
Transport media
D. Enriched media
Enriched media contain the nutrients required to support the growth of a
wide variety of organisms, including some of the more fastidious ones.
They are commonly used to harvest as many different types of microbes
as are present in the specimen. Blood agar is an enriched medium in
which nutritionally rich whole blood supplements the basic
nutrients. Chocolate agar is enriched with heat-treated blood (40-
45°C), which turns brown and gives the medium the color for which it
is named.
Media Preparation
1. Measure out approximately 90% of the final required volume of tissue
culture grade water (Product No.W 3500), e.g. 900 ml for a final
volume of 1000 ml. Select a container twice the size of the final
2. While stirring the water add the powdered medium and stir until completely
dissolved. Heating may be required to bring powders into solution.
3. Rinse the original container with a small volume of tissue culture grade
water to remove traces of the powder. Add to the solution in Step 2.
4. Add desired heat stable supplements (e.g. sucrose, gelling agent, vitamins,
auxins, cytokinins, etc.)
5. Add additional tissue culture grade water to bring the medium to the final
6. While stirring, adjust medium to desired pH using NaOH, HCl or KOH.
7. If a gelling agent is used, heat until the solution is clear.
8. Dispense the medium into the culture vessels before (or after) autoclaving
according to your application. Add heat labile constituents after
9. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C,
for the time period described under Sterilization of Media Protocol.
10. Allow medium to cool prior to use.

Review questions
List the steps you would go through to make tryptic soy agar slants.

What are the three main types (in terms of their physical forms) of
microbiological culture media?
____________________ ________________ ____________________


Section 2.1:- introduction

Microbial growth is the orderly increase in cellular constituents that

result in the formation of new cell. This process depends on
physiological capabilities of the species, the availability of nutrients,
and the environmental conditions such as pH, temperature, and osmotic
pressure. So to cultivate the microbes all the previous conditions must
be available.

Section 2.2: - Pure culture

A pure culture contains a single strain of microbe in which all
cells are derived from a single parent cell. In nature microbes exist in
mixed populations. For most purposes microbiologist must work with
pure culture to ensure that the phenomena they observe are attributable
to a given species.
The techniques for growing pure culture depend on physically
separating microbes in an environment that enable them to grow. This
technique was discovered by Koch.

Section 2.3: - Isolation techniques

A bacterium will grow into a distinct colony when it physically
isolated on suitable solid growth medium. Agar is used as a solidifying
agent for these media
1- The streak plate technique:
It is common for isolating pure colonies of bacteria and yeast
by streaking a sterile inoculating loop back and forth across the plate
until the organisms are physically separated on the agar surface. The
organisms grow into well-isolated


2- Pour plate

Pour plate can be made by repeated dilution of a bacterial culture

in a tubes of melted agar at about 47 to 48 C. The content of each tube
is poured into a petri dish and allowed to solidify.

Section 2.4: -Stock culture

Pure culture of microorganisms can be maintained in stock
culture for periods varying from weeks to years. Stock culture of
bacteria, fungi, and algae are maintained on slant (used for aerobes) or
in stabs (used for anaerobes) (figure 2.2). Stock culture prepared for
long-term storage are either freeze-dried or stored in liquid nitrogen at –
196 C or at –75 C in low temperature freezer.

Figure 2.2: agar slant and agar stab

- Review
What is the purpose of the spread-plate technique?

Make a drawing of the distribution of the colonies on each petri plate.

Session 6:
Collection of clinical specimens
Stool Specimen Collection
Stool specimen collection is the process of obtaining a sample of
a patient's feces for diagnosic purposes.
This procedure is used to test for infectious organisms, mucus,
fat, parasites, or blood in the stool.
Depending on the proposed analysis of the feces, watery feces
will not be suitable for conducting a test for any fat that may be
present, but can be used for other analyses, such as testing
for bacteria.
A stool specimen or culture can also be called a fecal specimen
or culture. A specimen of freshly passed feces of 1/2 to 1 ounce
(15 g to 30 g) is collected, without contamination of urine or toilet
tissue, into a small container that may have a small spoon or
spatula attached inside the lid of the cup for easier collection of
the sample.
Adult and older children patient can collect the specimen by
passing feces into plastic wrap stretched loosely over the toilet
bowl. A portion of the sample is then transferred into the supplied
With young children and infants wearing diapers, the diaper
should be lined with plastic wrap. A urine bag can be attached to
the child to ensure that the stool specimen is not contaminated
with urine.
For a bedridden patient, the specimen should be collected in a
bedpan lined with plastic wrap, and the nurse can transfer a
portion of the feces into the appropriate container.
Follow the manufacturer's guidelines if a commercial collection kit
is used.

If occult blood is suspected, the patient should be given a
mild laxative and should avoid eating foods rich in meat extracts
or leafy vegetables three days prior to the test. If the patient's
gums bleed when brushing their teeth, the mouth should be
cleansed with mouthwash and wiped with a cloth to avoid blood
entering the digestive system and contaminating the stool
Certain drugs may interfere with the analysis of the specimen,
and the patient should avoid ingesting products such
as antacids, oily foods and drugs, and antibiotics. Barium
sulfate should be excluded two weeks prior to the test, and
medical procedure dyes three weeks prior to the test.
If fat in the stool is suspected, the patient will also be asked to
collect the samples in pre-weighed airtight containers.
All feces passed in a 24-hour period are collected over two or
three days and sent daily for analysis.

Urine Specimen Collection

The urine specimen collection is a procedure used to obtain a
sample of urine from a patient for diagnostic tests.
The purpose of obtaining a urine sample is to test for any
abnormalities that may be present, such as bacteria, ketones, or
The skin of the genital area should be cleansed with a mild
disinfectant to prevent contamination of the urine specimen or
irritation of the delicate membranes of the area.
A urine specimen is sometimes called a clean-catch, urine
culture, or midstream specimen of urine, and is a method of
collecting a quantity of urine for testing.

The procedure and the reasons for it are explained to the patient.
Able patients may be allowed to collect the urine sample,
following the guidelines explained by the nurse.
Nurses who collect the urine sample should be sure to wash and
dry their hands carefully. The items required for the procedure
are as follows:
a sterile urine cup for children and adults
a sterile urine bag for infants
a bedpan or urinal for patients unable to use the toilet
sterile swabs
sterile towels
sterile gloves
For females, the area around the vulva is wiped and dried
thoroughly with the sterile swabs and towels, working from front
to back, with the nurse wearing sterile gloves. If the patient is
unable to use the toilet, the bedpan is placed beneath her. When
the urine begins to flow, the first part is allowed to pass into the
toilet or bedpan. Then the sterile container is placed in position
and filled with the mid-stream portion of the urine. The remainder
of the urine is then allowed to pass into the toilet or bedpan. The
lid is placed securely on the cup.
For males, the area around the penis and urethra is wiped and
dried thoroughly with the sterile swabs and towels, working from
front to back, with the nurse wearing sterile gloves. If the patient
is uncircumcised, the foreskin should be held back during the
complete procedure to prevent the skin contaminating the
sample. The patient then begins to pass urine into the toilet or a
urinal. Then, the sterile container is placed in position and filled
with the mid-stream portion of the urine, taking care that the
penis does not touch the sides of the container. The remainder of
the urine is then allowed to pass into the toilet or urinal. The lid is
placed securely on the cup.
For infants, the genitals are cleansed and dried thoroughly using
the sterile wipes and towels. A sterile urine collection bag is
placed over the area, with the adhesive tape firmly stuck onto the
baby's skin. A fresh diaper is put on the child over the collecting
bag and checked frequently for the child having passed urine into

the bag. When the specimen is obtained, it is poured into a
sterile container and sent immediately for testing.

Skin Specimen Collection

Scrapings and Swabs
In patients with suspected tinea or ringworm any ointments or
other local applications present should first be removed with an
alcohol wipe. Using a blunt scalpel, tweezers, or a bone curette,
firmly scrape the lesion, particularly at the advancing border. A
bone curette is safe and useful for collecting specimens from
babies, young children and awkward sites such as interdigital
spaces. If multiple lesions are present choose the most recent for
scrapings as old loose scale is often not satisfactory. Any small
vellus hairs when present within the lesions should be epilated.
The tops of any fresh vesicles should be removed as the fungus
is often plentiful in the roof of the vesicle.
In patients with suspected candidiasis the young "satellite"
lesions which have not undergone exfoliation are more likely to
yield positive results if they are present. Otherwise the advancing
scaly border should be scraped. When lesions in the flexures are
moist and very inflamed it is more satisfactory and less painful to
roll a moistened swab firmly over the surface.
In patients with suspected cutaneous manifestations of systemic
pathogens scrap the lesions with a bone curette or blunt scalpel
as for tinea. A biopsy may be required in some cases.
A. Skin scrapings:
1. Make a wet mount preparation in KOH for direct microscopy.
Note a Calcofluor stained mount may also be necessary.
2. Inoculate specimens onto Sabouraud's dextrose agar slopes
containing chloramphenicol and gentamicin, but NO
cycloheximide and incubate at 35C. Maintain cultures for 4
B. Skin swabs:
1. Smear swab onto heat sterilized glass slide for Gram stain.
2. Inoculate specimens onto Sabouraud's dextrose agar
containing chloramphenicol and gentamicin, but NO

cycloheximide and incubate at 35C. Maintain cultures for 4
3. Where secondary bacterial infection is suspected, and
separate swabs for routine bacteriology were not collected, the
swab should first be inoculated onto a blood agar plate, followed
by the Sabouraud's agar containing the antibiotics and then
placed into Brain Heart Infusion Broth. All cultures should be
incubated at 35C. Maintain cultures for 4 weeks.
Sputum Specimen Collection
Sputum specimen collection is a procedure designed to collect
expectorated secretions from a patient's respiratory tract.
Sputum is collected to be used as a laboratory specimen for the
isolation of organisms that might be causing abnormalities of the
respiratory tract.
This procedure should not be performed if the patient is unable to
take several deep breaths or cough deeply from the lungs.
When secretions from the respiratory tract are expectorated, the
secretions are called sputum. A sputum culture is a sample of
expectorated sputum.
Induced sputum is a procedure to assist patients who have
difficulty expectorating sputum. The patient inhales nebulized
saline to loosen the sputum. To collect an induced sputum
sample, the patient's mouth should be rinsed thoroughly
with water to reduce the amount of oral bacteria that are
normally present from contaminating the sputum. The patient
then inhales 20–30 ml of hypertonic saline from an ultrasonic
nebuliser. The sputum is loosened and collected in a sterile
sputum container.
The patient should be supervised during the collection of the
sputum to ensure the expectorated product has come from the
lungs rather than saliva from the oral cavity. The sample is best
taken first thing in the morning when the production of sputum is

To collect an expectorated sputum sample, the patient should
gargle and rinse out the mouth with water to reduce the amount
of oral bacteria that are normally present from contaminating the
sputum. The patient must take a deep breath and cough into a
sterile sputum container.
For a suspected common bacteria, one sputum sample may be
required. If the suspected infection is more complex, a sputum
sample may be required on three to five successive mornings.
If there is any difficulty in expectorating, the physician may
suggest the use of an inhalation, an expectorant,
or physiotherapy to aid in producing sputum for collection. The
sputum should be transferred to the laboratory within two hours
for analysis.

Sputum is mucoidal in appearance, resembling the thick liquid
secreted by the mucous glands. It can be clear, white, or
greenish in color, even blood stained. Blood in the sputum is
called haemoptysis and may be a pink froth, mucus with a streak
of blood, or an obvious clot, red in color representing fresh blood
or brownish representing old blood. Haemoptysis may indicate
that there has been some trauma to the respiratory tract, or that
there is an infection present such as tuberculosis or
even carcinoma. If it is determined that the blood is not from a
simple cut to the mouth or a nosebleed, it is considered a serious
condition and should be treated immediately. The sputum may
also be frothy, indicating that the patient's pulmonary blood
pressure is raised. Mucopurulent sputum contains mucus and
pus and indicates an infection, such as an abscess, is present.
There may be an unpleasant odor associated with sputum


SPECIMEN COLLECTION (the swabs will be combined for

• Label the VT or UTM tubes with the patient name, date of birth,
collection date, specimen site
(nose or throat)
Nasal swab (collected from the nasal septum, not just the
anterior nares)
1. Stand at the side of the patient
2. Ensure the patient’s head is resting against the wall
3. Place your hand on the patient’s forehead (non-dominant
hand) and the thumb at the tip
of the nose
4. Use a viral swab and insert the swab into the closest nostril
horizontally, approximately
2–3 cm
5. Place sideways pressure on the swab in order to collect cells
from the midline nasal
6. Rotate the swab twice (2 × 360° turns) collecting the epithelial
cells (not mucous)
7. Place swab into the labelled VT or UTM tube (if UTM, fully
insert the swab into the tube,
snap the swab at the breakpoint, discard the residual shaft) and
tighten the cap..
Throat swab
1. Stand at the side of the patient
1. Ensure the patient’s head is resting against the wall
2. Place your hand on the patient’s forehead (non-dominant
3. Ask the patient to open their mouth widely and say ‘argh’
4. Use a viral swab and insert the swab into mouth avoiding any
5. Place sideways pressure on the swab in order to collect cells
from the tonsillar fossa at

the side of the pharynx
6. Rotate the swab twice (2 × 360° turns) collecting the epithelial
cells (not mucous)
7. Place swab into the labelled VT or UTM tube (if UTM, fully
insert the swab into the tube,


Culture of the external auditory canal usually reflects the microbial
flora of the skin. The middle and
inner ear should be sterile.
There are several types of otitis externa. An acute localized infection in
the form of a pustule or furuncle
is due to Staphylococcus aureus. Erysipelas is caused by Group A strep
and involves the external ear
canal and soft tissue of the ear itself. Swimmer's ear is acute and diffuse
and related to maceration of the
ear from swimming or hot, humid weather. Gram negative bacilli,
particularly Pseudomonas aeruginosa,
are implicated. Chronic otitis externa is due to irritation from drainage
of the middle ear in patients with
chronic, suppurative otitis media and a perforated eardrum.
Most cases of acute otitis media occur in children, with pneumococcus
and Haemophilus influenzae the
most common isolates. Mastoiditis is a complication of chronic otitis
media and yields predominantly
anaerobic flora.
A. External Ear
1. Cleanse external ear with mild germicide to reduce contaminating
skin flora.
2. Any discharge in the external canal is a suitable specimen, for
aerobic culture only.
Sample discharge with an aerobic culturette, remove cap, place swab in
sleeve and
squeeze bottom of sleeve so that transport fluid from the sponge
moistens the swab.
B. Otitis Media

1. Tympanocentesis is a needle aspirate performed by a physician using
sterile equipment.
The bacteriological findings from children with acute otitis media have
been consistent
enough that there is little justification for routinely performing
tympanocentesis. The
procedure is usually reserved for patients with recurrent otitis media or
chronic persistent
otitis; and considered in neonate and the elderly since their etiologies
are unpredictable.
2. If the eardrum is ruptured, exudate should be collected by inserting a
sterile swab through
an auditory speculum.
C. Mastoiditis
An anaerobic culturette should be used to collect the specimen during
A. Transport aerobic and/or anaerobic culturettes to the lab ASAP.
B. If the volume of specimen obtained by needle aspirate is small, the
syringe, without needle, may
be transported to the lab immediately and hand delivered to the
microbiology staff.
C. As always, transport specimens to the lab in a biohazard bag.

Routine Microbiological Specimens

Physical Microscopic
Specimens Culture media
Ex. Exam
Wet film : Use standard loop of
- Pus cells with sterile tip of
- RBCs automatic pipette (10 µl)
- Trichomones - MacConkey agar
(MSU) / -Aspect
- Ova - Blood agar or CLED
morning -Color
- Epithelial cells Report colony count
sample -Bloody
- Monilia (CFU/ml):
(better) - Nitrite <102/ml not significant
>105/ml significant
Gram's Stain : bacterium

- if pus cells are 102-105/ml doubtful
numerous significant
Gram's stain : Routinely culture aerobic
- Pus cells - Blood agar with
- Candida staphaureus streak
- Borrelia vencenti or better incubate
swab - Fusiform bacilli anaerobic.
- Diphtheria - Add bacitracin dix
Wet film :
- Blood - if salivary reject
streaks sample - Blood agar (add
Sputum - purulent (epith. cells > 10/LPF, optalim/anaerobe)
(Morning - mucous Pus < 10/LPF) - Chocolate agar
Sample) - Viscid - MacConkey
- Salivary Gram's stain :
(rejected) - ZN : if
Gram's stain: - Blood agar (aerobic)
Wound - Pus + bacteria - MacConkey
swab or - If gas gangrene - Blood (anaerobic - on
pus report at one request for 5 days)
- LJ on request
Wet film :
- For trichomonas,
Vaginal pus, RBCs and
Culture immediately:
Gram's stain :
cervical - Prewarmed chocolate
- Vag. nugget score
swab agar + CO2
and clue cells
urethral - Blood agar (aerobic) &
- Urethral
discharge (anaerobic if request)
- PH
- KOH test

-Amount - Pre warmed chocolate +
secretion Wet film
-Failed CO2
and Gram's stain
massage - Blood agar (aerobic)
Ear and Gram's stain - Blood agar (aerobic)

Eye swab - pus + bacteria - MacConkey
- Blood agar
(anaerobic on
- Chocolate agar (ear)
- Sabouraud agar (on
From sediment :
- Color Cell number - Chocolate agar +
- Aspect - Uncentrifuged - dil CO2
- Blood 1:1 --- no X 5 /mm3 - Blood agar (aerobic
stained wet film & anaerobic / on
- Purulent Gram's stain request)
- Chemical Leishman (type of - MacConkey agar
ex. (on cells) - LJ
request) ZN - Sabouraud on
- Pre warmed
Cell count chocolate agar +
- Color
- mixed From deposit CO2 3 days
- Aspect
Wet film - Blood agar
- Blood
Gram's stain - MacConkey agar
CSF stained
Leishman - MacConkey agar
- Chemical
ZN anaerobic (on
ex. (on
*Keep CSF sample in request brain abces)
incubator - Sabouraud agar on
Wet film :
- MacConkey
- (pus, RBCs,
- Selenite Broth (18 h
Amoeba, cysts, larvae,
in 37C) then
Stool eggs,…..)
subculture on
MacConkey agar
Gram's stain
and XLD)
- (campylobacter)

-Make a drawing of the crystals or parasites of each microscopic field

References :

Microbiology by Richard harve and Pamela champ
LANGE by Jawetz, Melnick, & Adelberg
Microbiology concepts and applications by Paul A ketchum
Microbes in motion by Harry W seeley & john lee

Table of contents
Introduction and General safety
Session 1 Sterilization and Disinfection:
1) Heat
2) Moist heat
3) Autoclave:
4) Effect of disinfectant –Soap- on Bacteria (Hand Washing):
Session 2
1) Microscopy and bacterial staining
2) The light microscope
3) Microscopic measurement of organisms:
4) Applications of light microscope
1) Simple stain
2) Differential stains
3) Gram stain
4) Acid-fast stain
Session 4: Growth medium
1) Types of growth media
2) Minimal media
3) Selective media
4) Differential media
5) Enriched media
6) Media Preparation
Session5: Cultivation of bacteria
1) Pure culture
2) Isolation techniques
3) Pour plate
4) Stock culture
Session 6: Collection of clinical specimens
5) Stool Specimen Collection
6) Urine Specimen Collection
1) Skin Specimen Collection
2) Sputum Specimen Collection

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