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MICROBIOLOGY LABORATORY MANUAL

BASC 5205
Tri II

By:
Anjum Odhwani, MD MPH

1
TABLE OF CONTENTS
Microbiology Laboratory Instructions .

Supply List ..

Hand Washing .

Transmission of Disease ..

Microscopy ..

Aseptic Technique

Streak Plate Technique and Broth Characteristics

Preparation of a Bacterial Smear and the Simple Stain Technique .

Gram Stain

Acid Fast Stain .

Endospore Stain.

Motility

Negative Stain and Capsule Stain

Stethoscope and microorganism.

Biochemical Characteristics of Bacteria ..

Parasites and helminths

Anaerobes .

Microbiological Media..

The Staphylococci

The Streptococci ..

The Neisseria ..

The Enteric .

Preparation of yogurt

Physical Control of Microorganisms

Chemical Control of Bacteria ..


The Fungi .
MICROBIOLOGY LABORATORY INSTRUCTIONS

SAFETY: Living organisms will be used. Some cause disease while others do not. However,
any organism will cause disease under certain circumstances. Therefore, be sure to follow all the
instructions. Safety for the whole class depends on you.

GENERAL INSTRUCTIONS:
1. BRING only your lab manual, markers, colored pencils and lab coat to lab. LEAVE
personal belongings outside of the lab. There is no place to keep them in the lab.

2. WASH the table top with disinfectant before and after lab. (Antiseptic________,
Disinfectant__________)

3. WASH hands before and after the lab.

4. READ all lab instructions before starting an experiment.

PERSONAL ITEMS:
1. NO smoking, eating, drinking or gum chewing in the lab.

2. DO NOT touch fingers to your face, ears, eyes, etc. KEEP pencils out of your mouth.

3. TIE long hair back.

4. REPORT any open cuts or scratches to the instructor before working on an experiment.
USE antiseptic and bandage wounds. WASH hands well after the lab.

5. No open toed shoes or sandals.

DISCARDING INFECTIOUS MATERIALS:


1. DO NOT pour cultures into the sinks.
2. DISCARD all infectious disposable materials in buckets.
3. PUT all paper trash in the regular trash container unless it has become contaminated.
(Paper towels go in the regular trash.)
4. SPILLS of cultures should be COVERED with disinfectant and be REPORTED to the
instructor immediately. DO NOT attempt to clean up the spill unless you have been
instructed to do so.
SUPPLIES IN THE DRAWERS:

Staining Tray

2 Inoculating Loops Staining Tray


2 Inoculating Needles

Clothes Pin

Lens Paper
Inoculation loop and needle
Bibulous Paper

Forceps

Ruler Clothespin

Microscope Slides

Bunsen burner
Lens paper
Striker/Lighter

Bibulous paper

Bunsen Burner

Striker

Forceps

Ruler
Hand Washing

Objectives
Describe and demonstrate proper hand washing technique;
Describe the importance of proper hand washing technique in prevention of transmission of
pathogens.

Material
1. Sink
2. Paper Towel
3. Water
4. Soap

Procedure

1. Wet your hands with clean, running water (warm or cold), turn off the tap, and apply soap.
2. Lather your hands by rubbing them together with the soap. Be sure to lather the backs of
your hands, between your fingers, and under your nails.
3. Scrub your hands for at least 20 seconds.
4. Rinse your hands well under clean, running water.
5. Dry your hands using a clean towel or air dry them.
6. Turn off the water using the same paper towel to avoid recontamination of your clean
hands.

Microbiology Lab Manual


DEFINITIONS:
1. Epidemiology - study of the occurrence, distribution and spread of disease in humans.

2. Disease - any adverse internal condition severe enough to interfere with body functioning.

3. Infection Successful invasion of the body by pathogenic microorganism.

4. Reservoir Living or nonliving continuous source of infectious disease.

5. Carriers In human pathology, continuous asymptomatic human source of infection. rese


a. Chronic carriers - shed pathogens all their life.
b. Casual carriers (transient) - shed pathogens only temporarily and do not get the
disease.
c. Incubatory carriers - show no symptoms in the communicable period because they
are in the incubation phase of disease. They also shed organism temporarily.
d. Convalescent carrier - shows no sign of disease because they have recovered from
the disease. They can shed organism temporarily.

6. Vectors Any living organism that can carry pathogens from one host to another. The
most common vectors are the arthropods such as insects eg. Mosquito, fleas, lice
and ticks.
a. Biological vector Organism that transmits a pathogen and within which the
pathogen can multiply to high numbers.
b. Mechanical vector Organism such as fly that physically moves contaminated
material from one location to another.

7. Direct Transmission - pathogens are passed from one person to another by direct body
contact examples are
a. Skin to skin contact eg. Herpes labialis or cold sore
b. Sexually transmitted eg. Genital herpes, HIV, Gonorrhea etc.
c. Transplacental Transmission eg. Rubella, Toxoplasmosis and Syphilis
transmitted from mother to fetus.

8. Indirect Contact Spread of pathogens from one host to another via any of the following
methods.
a. Fomite Transmission- inanimate objects that transmit disease such as spoons,
cups, bedding, etc.
b. Droplet Transmission Spread of pathogens from one host to another via
respiratory droplets. People in close proximity can inhale those droplets, resulting
in disease. In droplet transmission a cloud of water droplets travel less than 1
meter.
c. Aerosol Transmission A cloud of water droplet can travel more than one
meter.
d. Fecal-oral transmission - usually from contaminated food or water sources

Microbiology Lab Manual


9. Probiotics- Live microorganisms which when administered in adequate amounts confer a
health benefit on the host.

10. Antiseptic- Chemical used to inhibit or kill microorganisms on living cells and tissues.

11. Disinfectant- Physical or chemical agents to inhibit or destroy microorganisms on inanimate


objects.

12. Sterilization- The process of destroying or removing all microorganisms and viruses
through physical or chemical means.

13. Disinfection- Process of reducing or eliminating pathogenic microorganisms or viruses in


or on a material so that they are no longer a hazard.

14. Pasteurization- Use of heat to kill pathogens and reduce the number of spoilage
microorganisms in food and beverages.

Microbiology Lab Manual


TRANSMISSION OF DISEASE

OBJECTIVES:
1. To observe how pathogens can be transmitted by touching.
2. To observe how transmission can be prevented.
3. To observe which method of washing hands is most effective in preventing disease
transmission.

MATERIALS:
1. Yeast suspension Fleishmans commercial yeast for baking (Brewers yeast), it is a
member of the genus Saccharomyces. Brewers yeast is considered non-pathogenic.
Please still use caution when handling this organism. If there is a possibility of an
allergic reaction please do not directly participate in this experiment but act as an
observer.
2. Sabourauds Dextrose agar has a relatively high sugar concentration (40g/L) and a low
pH of 5.6; therefore these conditions favor fungi over bacteria.
3. Hand sanitizer
4. Soap and water

PROCEDURE: Work in a group


1. Clear the work area and wash the bench with a disinfectant
2. Rinse the table with plain water twice to remove disinfectant residue.
3. Label three (3) Sabouraud s Dextrose agar plates (on the bottom where the agar is) with
your station number, your initials and as follows:
a. Plate 1 Paper towel
b. Plate 2 - Hand sanitizer
c. Plate 3 - Soap and water
4. Pour the yeast suspension on the cleared and rinsed area of the bench top. Both people
from one side of the table will share 1 tube of yeast.
5. With your lab partner to ensure that you do NOT drip yeast on the floor, others or other
objects in the lab.
6. One person place 4 fingers of one hand in the yeast suspension. Wipe the yeast off with a
dry paper towel. Now, touch your fingers GENTLY to the agar in the plate marked paper
towel. Dispose of the paper towel in the biohazard bucket. Wash your hands with soap
and water.
7. Place 4 fingers in the yeast again. GENTLY blot the excess yeast from your fingers onto
a paper towel. Use the hand sanitizer to clean your hands. Touch your fingers GENTLY
to the agar in the plate marked hand sanitizer. Wash your hands with soap and water.
8. Place 4 fingers in the yeast suspension. Wash your fingers with soap and water and
carefully blot your fingers with a paper towel. GENTLY touch your fingers to the agar
plate marked soap and water. Wash hands with soap and water thoroughly.
9. Place plates upside down in the carrier at the back of the room so they can be incubated at
25oC.
10. Clean the work station TWICE with disinfectant. Faculty will randomly sample work
station to see if they are clean.

Microbiology Lab Manual


RESULT:

1. Which plate is showing least amount of growth?

2. Which plate is showing maximum growth?

QUESTIONS

1. List different modes of transmission of disease causing organisms?

2. In your opinion, which is the most effective method to prevent disease transmission?

3. What is the difference between an antiseptic and a disinfectant?

Microbiology Lab Manual


ASEPTIC TECHNIQUE
OBJECTIVES:

1. Describe aseptic technique and its importance.


2. Demonstrate aseptic technique
3. Describe different culture media

DEFINITIONS:
1. Septic: characterized by the presence of pathogenic microbes in living tissue.

2. Aseptic: characterized by the absence of pathogenic microbes.

3. Aseptic Technique: methods and procedures used in the laboratory to prevent


contamination of the work area, the worker and the cultures with microorganisms.

4. Broth Medium: a solution (liquid) containing nutrients required by microorganisms for


growth.

5. Agar: a seaweed polysaccharide containing D and L galactose; agar makes broth solid.
Microorganisms cannot digest agar, therefore, the medium remains hard.

6. Solid Medium: broth that has agar in it to make it hard.

7. Petri Plates: flat plastic plates with a lid; usually filled with a solid medium.

DEMONSTRATION:

1. Wash area with disinfectant.


2. Set up area with all of the equipment and media needed.
3. Light the Bunsen burner. Set the flame so that there is a blue cone within the flame. The
hottest part of the flame is just below the tip of the blue cone.
4. Label all tubes and Petri plates. Label Petri plates on the bottom (not the lid). Put your
station number, and organism or source of the sample. Label test tubes lid with station
number and microorganism on glass tube.
5. Wash hands.
6. Inoculate the media.
1. broth to broth
2. broth to plate
7. Incubate the samples at the appropriate temperature.
8. Properly discard items used.
9. Wash the area with disinfectant again.

Microbiology Lab Manual,


BROTH CHARACTERISTICS AND FOUR QUADRANT STREAK PLATE
TECHNIQUE

OBJECTIVES:
Demonstrate aseptic inoculation of broth medium.
Describe growth characteristic in broth
Define pure culture and a bacterial colony
Explain the purpose of a four quadrant streak plate.
Demonstrate how to make a four quadrant streak plate.
Describe the characteristics of an isolated colony (form, margin and elevation)

Bacteria have growth characteristics in broth cultures. After inoculation of broth media, bacteria
may exhibit a particular form of growth. These include the formation of a thin surface film called
pellicle (obligate aerobe), accumulation of the cells at the bottom called sediment (obligate
anaerobe and facultative anaerobe), and clouding of the medium called turbidity (facultative
anaerobe).

Bacteria are found in mixed cultures in many environments like food, soil, water, and the human
body. A pure culture must be obtained before the characteristics of the organism can be studied.
A pure culture is a culture which has a single type of organism. In this exercise you will learn
the most common technique for isolating organisms from each other. The purpose of four
quadrant streak plate is to isolate a bacterial colony. In the streak plate method, the mixture is
thinned out on an agar plate so that individual organisms are separated from each other. Each
organism forms a colony. A colony is an aggregation of cells arising from a single parent cell. A
colony of organism that comes from a single organism is a pure culture because all the organisms
will be genetically identical. In a colony there are one million bacteria. The pure colony can be
further cultured and characterized.

The characteristics of a bacterial colony are determined by the genetic makeup of the cell.
Bacterial colonies have a particular
Form (shape)
Margin (edge)
Elevation (Raised, convex, umbonate)
Size (punctiform, small, moderate and large)
Appearance glistening or dull
Pigmentation nonpigmented (cream, tan, white) pigmented (purple, red, yellow)
Optical characteristic (opaque, translucent, transparent) of the colony depending on the
bacterial type.
You will observe the colony types produced by the bacteria found in a mixture.

MATERIALS:
1. nutrient agar plates (TSA) - one per person
2. nutrient broth tubes (TSB) or (BHI) - 4 tubes one per person
3. inoculating loop
4. pure bacterial cultures - Serratia, B. subtilis, E. coli, Staph. epidermidis.
5. a mixture of organisms - (Serratia, E. coli, Staph. epidermidis)

Microbiology Lab Manual,


PROCEDURES:

Broth Cultures - Work individually


1. Using a wire loop inoculate nutrient broth cultures with selected organisms.
2. Place one type of organism in each separate tube.
3. Place your tubes in the appropriate carrier.
5. Leave the caps loose.
6. DO NOT SHAKE THE TUBES.
7. Observe the broth growth characteristics.

Streak Plate Technique - Work individually


1. Each student will take one nutrient agar plate and label the bottom of a nutrient agar
plate with your station number and microorganism.
2. Aseptically take a loop full of the pure culture from the tube.
3. Skate the loop back and forth across one section of the plate.
4. Be careful not to overlap streak marks.
5. Sterilize the loop to remove any excess bacteria.
6. Cool the loop by touching it to the edge of the agar in an area that is still free of
organisms.
7. Go back into the streaked area with the loop and pull some of the organisms out onto
a new section of the plate.
8. Repeat steps 4 - 8.
9. Continue streaking out the organisms until 4 sections of the plate have been streaked.
10. Be sure to sterilize the loop between sections on the plate.
11. Place the plates upside down. The plates will be incubated for 24-48 hours.
13. Observe the plates for isolated colonies and the colony characteristics.

Microbiology Lab Manual,


Streak plate for isolation technique

Quadrant 1 - Apply a
loop full of culture Quadrant 2

Flame to
start
Flame
loop
1
2

Flame loop

Quadrant 3
Quadrant 4

Flaming 4
optional

Microbiology Lab Manual,


BROTH CHARACTERISTICS

370 C

Serratia turbid + red

B. subtilis pellicle

Staph. epidermidis sediment?/no growth?

E. coli Fine turbidity

STREAK PLATE
370 C

Staph. epidermidis F - circular


M - entire
E - convex
C - white

E. coli F - irregular
M - undulate
E - umbonate
C - cream

Serratia F - circular
M - entire
E - umbonate
C - red

B. subtilis F - irregular
M - Lobate
E - flat
C - cream

Mix culture

Microbiology Lab Manual,


Questions
1. What is the importance of an aseptic technique?

2. Describe a broth medium.

3. Where do you label a Petri plate?

4. What information do you put on a test tube or a Petri plate?

5. What is the name of solidifying agent used most successfully in bacterial nutrient
media?

6. What is the purpose of streak plate?


Microbiology Lab Manual,
7. How colony characteristics are described?

8. How growth characteristics in a broth are described?

9. A colony consists of how many bacteria?

RESULTS and OBSERVATIONS

Broth Characteristics

Microorganism Broth characteristics

Staphylococcus epidermidis

Bacillus subtilis

Serratia marcescens

E. coli

Microbiology Lab Manual,


Streak Plate colony characteristics

Microorganism Form Elevation Margin

Staphylococcus
epidermidis

Bacillus subtilis

Serratia marcescens

E. coli

Microbiology Lab Manual,


MICROSCOPY

OBJECTIVES:

1. Explain the difference between a simple and compound microscope and be able to name
some of the other types of scopes currently in use today.
2. Identify various parts of a compound microscope including the: condenser, diaphragm,
course and fine focus adjustments, objectives, revolving nosepiece and rheostat.
3. Define resolving power, total magnification, parfocal, working distance, real image, and
virtual image.
4. Demonstrate how to use and clean a microscope.

Historically, Anton van Leeuwenhoek is credited with initiating the use of the microscope as a
research tool. Using a simple microscope, he described many bacteria and protozoa in 1674.
The single lens, simple microscope was impractical due to the limitations in magnification and
resolution. The compound microscope with a double lens system was developed over the next
two centuries. The compound microscope that we know today, with a sub-stage condenser and
oil immersion lens, was designed by 1866. Refinements since then have made the light
microscope the powerful tool it is for the observation of microorganisms. The optics are
excellent, and the microscopes are almost mechanically flawless. The phase contrast, the
fluorescence and the electron microscopes have been developed since World War II.

The microscope is a quality tool, but it is useless without knowledge of how to achieve the
maximum resolution, the greatest magnification and other tricks of the trade. The resolution or
the sharpness of the image depends on the adjustment of the diaphragm, condenser and the use of
immersion oil. Haphazard adjustments will cause distortions in the image. This lab is designed
to familiarize you with the strengths and weaknesses of your microscope. A definition sheet has
been prepared to help you understand this instrument.

Microbiology Lab Manual,


Microbiology Lab Manual,
MICROSCOPY DEFINITIONS

1. Simple Microscope: one lens.


2. Compound Microscope: two lenses -- Ocular (closest to the eye) 10X
Objective (closest to the specimen) 4X low,
10X, 40X, 100X high
3. Condenser: group of lenses below the stage which functions as a light gathering system
which sends light to the specimen from the light source.
4. Diaphragm: iris-like closure system below the stage which regulates the amount of light
passing through the condenser.
5. Resolving Power: the ability to distinguish 2 points as distinct, separate objects rather than
as one blurred image. Under oil immersion (100X) this distance between points should be
0 .22m. The resolving power depends on the wave length of light (shorter = better), design
of the condenser and use of oil immersion.
6. Numerical Aperture: relates to the size of the cone of light entering the objective and the
medium surrounding the objective (usually air).
7. Immersion Oil: prevents loss of light rays due to diffraction because the oil has the same
refractive index as glass and improves resolution.
8. Total Magnification: ocular magnification multiplied by the objective magnification.
(example: 10X times 100X = 1000X total magnification).
9. Parfocal: once the specimen has been focused under low power (10X) the microscope is
parfocal if you are able to switch to a higher magnification with a minimum of focal
adjustment.
10. Working Distance: distance between the bottom of the objective lens and the slide. As the
magnification increases, the working distance decreases.
11. Light Intensity: less light is required at low magnifications. As the magnification increases,
the need for light increases. However, too much light can "burn out" the image.
12. Diameter of Field: as the magnification increases the diameter of the field decreases.
13. Real Image: image passing into the objective lens from the specimen.
14. Virtual Image: real image is further magnified by the ocular lens and passes to the retina of
the eye. Virtual image is upside down and reversed left to right.
15. Coarse Focus: used to bring the specimen into approximate focus.
16. Fine Focus: used to bring the specimen into clear focus.
17. Rheostat: electrical adjustment for the light bulb.

Questions
1. How is the total magnification of a microscope determined?

2. What is the purpose of the immersion oil?

3. Define resolving power.

4. Is your microscope parfocal or nearly so? How can you tell?

5. Who invented microscope?

Microbiology Lab Manual,


PREPARATION OF A BACTERIAL SMEAR AND THE SIMPLE STAIN TECHNIQUE

OBJECTIVES:

1. List the steps of making a bacterial smear


2. Demonstrate how to make a bacterial smear aseptically.
3. Explain why to air dry a bacterial smear prior to heat fixing and the purpose of air drying a
slide.
4. Explain the purpose of a simple stain
5. Differentiate between different sizes, shapes and arrangements of bacteria
6. List basic dyes which can be used to make a simple stain and how they work in staining the
organism.

Some of the criteria for classifying or identifying bacteria are the shape, size and arrangement of
the bacteria. These characteristics can be visualized in the microscope when the organisms are
applied to a slide and are stained. The three basic shapes are cocci, bacilli and spirals. This
exercise will demonstrate the application of bacteria to a slide, the simple stain procedure and the
handling of pure bacterial cultures.

The cultures you use will be pure cultures which contain a single type of organism. The cells
may be grown in liquid nutrient broth or on a solid nutrient agar. The agar used will be in slants
because the bacteria are easier to pick up from a slanted surface than from the surface of a deep.

The bacteria will be transferred to glass slides by aseptic (sterile) technique. This technique
insures that the cultures remain pure and that the environment is not contaminated. The bacteria
must be fixed to the slide so that the bacteria will not wash off during the staining procedure.
A bacterial smear is the fixed bacteria on a slide that are ready for staining.

When a simple stain is made, a single dye is used. Various dyes can be used. The only
requirement is that the dye must be a basic dye with a positive (+) charge, so that it will be
attracted to the negatively charged cytoplasm of the bacterial cell. Crystal violet, methylene
blue, safranin, and malachite green are positively charged basic dyes.

Microbiology Lab Manual,


Bacteria display a large diversity of cell morphologies and arrangements

Microbiology Lab Manual,


MATERIALS:
1. pure bacterial cultures - Bacillus subtilis and Staphylococcus epidermidis.
2. microscope slides
3. basic dyes such as crystal violet, methylene blue, malachite green, and safranin
4. bibulous paper
5. distilled water
6. staining tray
7. inoculating loops
8. Bunsen burner

PROCEDURE: Work in groups of two.

Preparation of a bacterial smear from an agar culture.


1. Label slides immediately with organism name of the organism.
2. Make a large puddle of distilled water on a spare clean slide. Use this as a source of
water needed to make smears.
3. Using aseptic technique, add a small drop of distilled water directly to center of slide
using a loop.
4. Using aseptic technique, remove bacteria from slant.
5. Mix bacteria in water, distribute mixture in a thin film over the surface of the slide.
6. Allow mixture to TOTALLY AIR DRY.
7. Heat fix slide by passing it (organism side up) through the flame of the Bunsen burner
3-5 times.
8. Slide is now ready to be stained.

Preparation of a simple stain. Work in groups of 2 (two).


1. Place an air dried, heat fixed bacterial smear on a staining tray. Use an air dried, heat
fixed smear of Bacillus subtilis and Staphylococcus epidermidis.
2. Cover Bacillus subtilis smear with methylene blue and Staphylococcus epidermidis
with crystal violet.
3. Leave the dye on for one minute.
4. Wash the dye off with water.
5. Blot the slide dry with bibulous paper. DO NOT RUB.
6. Examine the slide under oil emersion lens.
7. The shape, color and arrangement can only be clearly seen on 100X oil immersion.
Place one drop of oil directly on the smear.
8. To remove the oil from the slide (after you have observed the stained smear), let it
drain off onto a paper towel. DO NOT RUB OIL OFF.

Microbiology Lab Manual,


RESULTS and OBSERVATIONS
Simple Stain

Microorganism Shape Arrangement

Staphylococcus epidermidis

Bacillus subtilis

Make Drawings

Microbiology Lab Manual,


GRAM STAIN

OBJECTIVES:

1. Discuss the purpose and mechanism of Gram stain.


2. Compare the difference between Gram (+) and Gram (-) cell walls.
3. List the steps of Gram stain.
4. List the dyes used in Gram stain
5. Recognize a Gram (+) organism from a Gram (-) organism under microscope.
5. Discuss the importance Gram stain in diagnostic clinical microbiology.

Hans Christian Gram developed a method of staining bacteria in 1874. He found that some
bacteria would retain their color after treatment with alcohol while others would lose the color.
The microorganisms could be divided up into two groups based on the results of this staining
procedure. The organisms that retain crystal violet stain are called Gram positive. Those that
lose crystal violet stain and retain safranin are called Gram negative. The (+) and (-) do not refer
to the electrical charge; they simply refer to the response of the organism to the staining
procedure. The Gram stain is still the single most important procedure in Microbiology because it
is the first biochemical step in the identification of organisms after an isolated colony is obtained.
Biochemically, the Gram stain works because of differences in the cell walls of bacteria.
Gram positive organisms have multiple layers of peptidoglycan in the cell wall.
Primary Stain
Crystal Violet is used first and stains all cells purple.
Mordent
Grams Iodine serves as a mordent, a substance that increases the cells affinity for a stain. It
binds to primary stain, thus forming an insoluble complex. The resultant crystal-violet-iodine
complex serves to intensify the color of the stain.
Decolorizing Agent
Acetone- Alcohol cannot easily remove the dye from the peptidoglycan. Its action is determined
by the concentration of lipids and the thickness of the peptidoglycan layer in bacterial cell walls.
In Gram negative cells the alcohol increases the porosity of the cell wall by dissolving the lipids
in the outer layers. Thus crystal violet iodine complex is easily removed from the thin layer of
peptidoglycan. The thick layer of peptidoglycan in gram positive bacteria will retain crystal violet
iodine complex.
Counterstain
Safranin is the final stain used to stain cells that have been decolorized. Since only Gram-negative
cells undergo decolonization, they will now absorb counterstain and will be stained red or pink.

Gram Stain

Microbiology Lab Manual,


MATERIALS:
1. pure bacterial cultures (Staphylococcus epidermidis and Pseudomonas)
2. slides
3. inoculating loop
4. Bunsen burner
5. crystal violet
6. Gram's iodine
7. acetone - alcohol decolorizer
8. safranin
9. bibulous paper

PROCEDURE: Work in groups of 2 (two).


1. Cultures needed are Staphylococcus epidermidis and Pseudomonas.
2. Place air dried, heat fixed smear of on a staining tray.
3. Cover the smear with crystal violet (primary stain) for one minute.
4. Wash with a light stream of water.
5. Cover the smear with Gram's iodine (mordant) for one minute.
6. Wash with a light stream of water.
7. Cover the smear with decolorizer for 15 sec or let the decolorizer run over the smear
until no more purple dye runs off.
8. Wash with a light stream of water.
9. Blot dry the slide with bibulous paper.
10. Cover the smear with safranin (counterstain) for 2 minutes.
11. Wash with a light stream of water.
12. Blot slide dry with bibulous paper.
13. Examine the slide under oil emersion lens.

STEP GRAM (+) GRAM (-)


Primary stain - purple purple
crystal violet

Mordant- purple-blue purple-blue


iodine

Decolorizer - purple clear, colorless


alcohol-acetone

Counterstain - purple orange-red, pink


Safranin
GRAM (+) GRAM (-)
1. 30 or more layers of peptidoglycan 1. 2 to 3 layers of peptidoglycan
2. No outer layer 2. Outer layer Lipopolysaccharide (LPS)
3. more susceptible to lysozyme 3. less susceptible to lysozyme
Penicillin and basic dyes penicillin and basic dyes
4. From cytoplasm secrets exotoxin 4. Cell wall is endotoxin
5. Alcohol will not remove 5. Alcohol removes lipid layers
dye embedded in the with dye molecules, any dye
peptidoglycan that reached the peptidoglycan
leaks out. When the lipid washes
off the dye washes off, therefore
no purple color
Microbiology Lab Manual,
RESULTS and OBSERVATIONS
Gram Stain

Microorganism Gram positive or Gram Color Shape


negative

Make Drawing

Microbiology Lab Manual,


Questions

1. Why do you air dry a bacterial smear?

2. Why is it necessary to heat-fix a bacterial smear?

3. Describe the characteristic of a basic dye?

4. List four basic dyes

5. Who invented Gram stain?

6. Based upon the result of Gram Stain, bacteria are divided into how many groups?

7. Describe the cell wall of Gram positive and Gram negative bacteria?
a. Gram positive b. Gram negative

8. Under the microscope, Gram positive bacteria are of which color and gram negative
bacteria are of which color?

Microbiology Lab Manual,


ENDOSPORE STAIN
OBJECTIVES
1. List two (2) genera of bacteria that produce endospores and name diseases caused by spore-
forming bacteria.
2. Explain what is present in endospore coat and why they are resistant to staining.
3. List the dyes used in endospore stain.
4. Demonstrate the steps of endospore stain.
5. Identify endospores under microscope.

Endospores are heat resistant dormant form of bacteria. The genus Bacillus and Clostridium are
groups of organisms that produce endospores. The endospore is made up of many layers
including the spore coat and outer layer spore crust. It is the spore crust which protects the
bacteria from heat, drying and chemical agents including some antibiotics.
The endospore coat and core are stabilized with dipicolinic acid. Some of the diseases that are
caused by endospore-forming bacteria are Clostridium tetani causes tetanus, Clostridium
botulinum causes botulism, Clostridium perfringens causes gas gangrene and Bacillus anthracis
causes anthrax. Endospores can be visualized using the endospore stain.

Endospore Stain

MATERIALS:
Endospore Stain
1. Endospore producing organism - (Bacillus subtilis)
2. malachite green
3. safranin
4. steaming apparatus (Bunsen burner, ring stand, beaker of water and a wire gauze)
5. slides
6. bibulous paper

PROCEDURE: Work in groups of 2 (two).


Endospore Stain
1. Place a heat fixed bacterial smear on the staining tray.
2. Place the slide on the wire gauze over the steaming apparatus. (Make sure the water
is boiling slowly).
3. Place the bibulous paper (cut to size) over the dye.
4. Cover the smear with malachite green (primary stain).
5. Steam the slide for 5 minutes.
6. Don't let the slide dry out. Add dye as needed to keep the paper wet.
Microbiology Lab Manual,
7. Wash the slide with water.
8. Blot dry the slide with bibulous paper.
9. Cover the smear with safranin (counterstain) for 2 min.
10. Wash the slide with water.
11. Blot the slide with bibulous paper.
12. Observe the slide under oil emersion lens.

Endospore Stain
Sketch your results here

Endospore Stain

Microorganism Shape Endospore present or absent

1. What are Endospores?

2. Name two genera which make endospores.

3. What is the name of the chemical present in the spore- coat and core that stabilizes
endospores?

4. List three bacteria and diseases caused by endospore-forming bacteria.


a.
b.
c.

Microbiology Lab Manual,


ACID FAST STAIN

OBJECTIVES:
1. Identify the biochemical difference between the cell wall of genus Mycobacterium and all
other bacteria
2. List the dyes used in Acid-fast stain
3. Demonstrate the steps of Acid-fast stain.
4. To recognize a Mycobacterium species from any other genus of bacteria.
5. Discuss the importance of an Acid-fast stain as a diagnostic test.

Some microorganisms particularly the members of the genus Mycobacterium has cell wall with a
high mycolic acid content and is visualized much better by acid-fast stain. Mycolic acid is a
group of branched chain hydroxy lipids. Mycolic acid prevents the cells from staining easily
with simple stains. Heating the specimen with carbolfuchsin (primary stain) and phenol allows
this red dye to penetrate the mycolic acid. Once the dye has penetrated the cell wall it cannot be
easily removed even with acidified alcohol. Hence, the organisms that stain with carbolfuchsin
that cannot be removed by acid-alcohol are said to be acid fast. Non-acid fast organisms have
the primary stain removed with acid-alcohol and can be counterstained with methylene blue.
Acid fast organisms are red while non-acid fast organisms are blue. This method is the Ziehl-
Neelsen method for the acid fast stain. Acid-fast stain is a differential stain.
Mycobacterium tuberculosis causes tuberculosis and M. leprae causes leprosy, acid-fast stain is
used as a diagnostic procedure for tuberculosis and leprosy.

Acid-Fast Stain

MATERIALS:
1. selected Mycobacterium species (Mycobacterium smegmatis)
2. non-acid fast organism (Staphylococcus epidermidis)
3. Ziehl-Neelsen carbolfuchsin
4. acid-alcohol
5. methylene blue
6. steaming apparatus
7. inoculating loop
8. scissor
PROCEDURE: Work in groups of 2 (two).
1. Set up the steaming apparatus; make sure the water is boiling slowly.
Microbiology Lab Manual,
2. Place a heat fixed bacterial smear of Mycobacterium smegmatis and Staphylococcus
epidermidis as a mixture on the steaming apparatus.
3. Cut bibulous paper to fit over the smear and place it on the slide.
4. Add carbolfuchsin (primary stain) to the paper on the smear.
5. Steam the slide for 5 minutes on the wire gauze of the steaming apparatus. Add dye
as needed to keep the paper wet during the steaming.
6. Wash the slide with water.
7. Add acid-alcohol decolorizer in drop wise fashion until the red dye stops running off
(or add acid-alcohol to the slide for 15 seconds).
8. Wash the slide with water.
9. Blot dry slide with bibulous paper.
10. Add methylene blue (counterstain) for 1 min.
11. Wash the slide with water.
12. Blot the slide dry with bibulous paper.
13. Observe the slide with 100X oil immersion.

RESULTS and OBSERVATIONS


Acid-fast Stain

Microorganism Acid-fast Stain Color Shape

Make Drawing

Questions
1. Name the genus which can be stained by acid-fast stain.

Microbiology Lab Manual


2. Acid-fast stain is a simple or differential stain.

3. Name the bacteria and diseases caused by acid-fast bacteria.


a.
b.

4. Name the primary and counter stain in acid-fast stain.

5. What is the name of decolorizer used in acid-fast stain?

6. Which long chain fatty acid is present in the cell wall of genus Mycobacterium?

MOTILITY

OBJECTIVES:

Microbiology Lab Manual


1. Discuss the purpose of flagella and list three (3) methods for testing for the presence of
flagella.
2. Describe how motility media differs from regular media.
3. Demonstrate proper inoculation of motility media.
4. Recognize growth characteristics in motility media and be able to interpret tubes as positive
or negative for motility.
5. Identify different flagellar arrangement.

Bacteria are complex organisms with a variety of structures. Flagella are used by organisms for
locomotion. Motility and the arrangement of flagella around the cell are important taxonomic
characteristics that are useful in characterizing bacteria. Flagella are long and thin that they are
hard to see.

Motility Test Medium (MTM)


This is a nutrient medium which is semi-solid. It is softer than regular agar so that the organism
can swim through it. Motility test medium with triphenyltetrazolium chloride provides an easy
method for determining motility. TTC in its oxidized form is colorless. As bacteria grow in the
presence of TTC, the dye is absorbed into the bacterial cells where it is reduced to the insoluble
red-colored pigment. Growth is indicated by the presence of the red color, and as motility
occurs, small to very large regions of color can be observed around the area of inoculation.
The medium is inoculated with a straight wire. If the organism is motile, it will swim away from
the line of inoculation into the surrounding medium. Nonmotile bacteria will grow along stab
line. In this test the flagella are not actually seen but the test is testing for flagella.

Flagella Stain
This is the direct method which shows the presence of flagella. Flagella stain builds up dye
around the flagella until it is large enough in diameter to be seen with a microscope.

A-Monotrichous B-Lophotrichous
C-Amphitrichous D-Peritrichous
MATERIALS:
Motility
1. two organisms based upon availability any organism can be used Proteus vulgaris, E.
coli, Staphylococcus epidermidis, Streptococcus pyogenes

Microbiology Lab Manual


2. motility medium (soft agar MTM) - 1 tube/group
Soft agar is nutrient agar that has about half the agar concentration of regular agar.
SIM is soft agar that has additional chemical components which make this medium
differential. This agar can also be used to determine hydrogen sulfide production and
indole production.
3. straight wire
4. prepared slides

PROCEDURE: Work in groups of 2 (two).


Motility
1. Label MTM tubes with station number and microorganism.
2. One group stabs one motility medium with one organism.
3. Other group stabs the other motility medium with the other organism.
4. Keep the caps of the tubes loose.
5. Place the tubes in the test tube rack. The tubes will be incubated at 370 C.
6. Observe prepared slides of flagella stain.
7. Observe the tubes and determine which organism has flagella and is motile after 48
hours.

RESULTS and OBSERVATIONS


Motility Test

Non-motile Motile
Questions
1. Name the structure used for motility in both eukaryotes and prokaryotes.

2. Describe different flagellar arrangements


NEGATIVE STAIN AND CAPSULE STAIN

OBJECTIVES:

Microbiology Lab Manual


1. Discuss the purpose of capsule in a bacterium.
2. Discuss the purpose of negative and capsule stain.
3. List 3 dyes used to make a negative stain and their mechanism of action.
4. To explain why you do NOT heat fix a negative or capsule stain.
5. Compare and contrast negative and capsule stain
6. Distinguish between negative and capsule stain under microscope.

The negative and capsule staining procedure is frequently used to examine capsule of bacteria.
Negative stain gets its name for two reasons. First, the background is stained instead of the
organism so the field of view looks like a photographic negative with light objects on a dark
background. Second, the stain used is an acidic dye with a negative charge that is repelled by the
negative cytoplasm of the bacterial cell.
Three stains are used for negative stain are Nigrosin, Congo red and India ink. Nigrosin and
Congo red are two acidic dyes. India ink is a neutral dye composed of particles too large to enter
a cell and does not have any charge on it. The ink fills in the background around the organism.
The background is dark while the cell is light. This reaction is not based on electrical charge,
just on particle size.

Negative Stain Capsule Stain

The capsule is a protective coating of the bacterial cell. It helps bacteria stick together and resist
phagocytosis. In capsule stain negative and simple stain procedures are combined to observe the
capsules. Typically an acidic stain such as Congo red or nigrosin, which stains the background,
and a basic stain which stains the bacteria are used in combination. The background will be dark
because of the negative stain and the organism will take basic stain. The capsule remains clear
since neither the acidic or basic stain can bind to capsule and appears as a white halo between the
bacteria and colored background.

The preparations for the negative stain and the capsule stains are very fragile. Negative staining
requires the use of either acidic dye or India ink. Bacteria are NOT heat fixed in this staining
technique as capsule is destroyed heat. Extra care must be taken to handle slides so that the
bacteria and stain do not wash off. Also, the bacteria are still alive so they can be infectious.
Diseases caused by encapsulated bacteria are S. pneumoniae causes pneumonia, otitis media and
meningitis, E. coli causes UTI, travelers diarrhea, meningitis and gastroenteritis, K. pneumoniae

Microbiology Lab Manual


causes pneumonia and N. meningitidis causes meningitis

Questions
1. When might one choose to perform the negative stain?

2. Can any stain be used for negative staining?

3. List the names of negative stains.


a.
b.
c.

4. What is the difference between negative and capsule stain?

5. Name the bacteria and diseases caused by the bacteria which possess capsule.

Microbiology Lab Manual


STETHOSCOPE and MICROORGANISM

OBJECTIVE

1. Test the stethoscope diaphragm for the presence of bacteria.


2. Test the effectiveness of alcohol swabs in disinfecting stethoscope diaphragm.
INTRODUCTION:
Stethoscope is a universal tool used in the clinics and hospitals. The same stethoscope may be
used on multiple patients thereby becoming a vector in the transmission of disease. Tryptic soy
agar can be used to test for the presence of bacteria on the diaphragm of the stethoscope. After
inoculation and incubation, if bacterial colonies are present on tryptic soy agar we can conclude
that there was bacterial contamination on the stethoscope diaphragm. In this experiment we will
test to see if alcohol is an effective method for disinfecting the stethoscope
MATERIAL:
1. Stethoscope
2. Two tryptic soy agar plates
3. Alcohol wipes
4. Bunsen burner

PROCEDURE: Work as a group

1. Label tryptic soy agar plates with station number and also label pretest on the 1st plate and

posttest on the 2nd plate.

2. Clean the diaphragm of the stethoscope with an alcohol wipe.

3. Let it air dry.

4. Gently press the diaphragm to the surface of the tryptic soy agar marked as pre-test.

5. Clean the diaphragm with an alcohol wipe

6. Use diaphragm of the stethoscope to listen for breath (heart) sounds on your partner.

7. Gently press the diaphragm to the surface of the tryptic soy agar plate marked as post-test.

Microbiology Lab Manual


8. Clean the diaphragm with an alcohol wipe.

9. Incubate the agar plates for 24-48 hours at 370 C and read the results.

RESULT:

Tryptic soy agar

Pre Test growth

Post Test growth

Questions:

1. What is a fomite?

2. What is the difference between sterilization and disinfection?

3. Are you sterilizing the stethoscope diaphragm when you clean it with an alcohol wipe?

Microbiology Lab Manual


BIOCHEMICAL CHARACTERISTICS OF BACTERIA

OBJECTIVES:

1. Discuss different biochemical processes occurring in bacteria and how these processes can
be used to identify bacteria.
2. Discuss the purpose and interpretation of each biochemical test.
3. Discuss substrate, enzyme and end products in each biochemical test.
5. Discuss the importance of positive and negative test results.

The staining procedures that were done in the previous labs allowed us to see some general
characteristics of bacteria. The size, shape and arrangement gave some hints about the type of
bacteria with which we were working. The Gram stain was the first step in classification.
However, it is impossible to identify a suspected pathogen without knowing the biochemical
characteristics of the bacterium.

The biochemical characteristics of a bacterium reflect the metabolic reactions occurring within
the cell. Each type of bacterium has its own set of biochemical reactions that are typical for that
organism. Therefore, organisms can be differentiated by biochemical reactions and classified
down to genus and species. There are many substrates that are utilized releasing products that
can be analyzed. We will be inoculating a variety of media with organisms to look at some of
the biochemical reactions for common organisms. The substrates, enzymes and products are to
be studied.

CARBOHYDRATE FERMENTATION

Fermentation is an anaerobic process. In fermentation, substrate such as carbohydrates


undergoes anaerobic dissimilation and produces an organic acid (lactic acid, formic or acetic
acid) that may be accompanied by gases such as hydrogen or carbon dioxide. The type of
carbohydrate fermented depends on the type of bacterium tested. The end products of
fermentation are usually acid and gas. Bacteria may produce acid and gas, acid alone, or neither.
We will look at the fermentation of a variety of sugars. The medium is nutrient broth
supplemented with a fermentable carbohydrate. The medium also contains the pH indicator,
phenol red which is red at neutral pH (7) and changes to yellow at a slightly acidic pH of 6.8. A
small inverted tube called Durham tube is also present in the broth for the detection of gas
production.
Fermentation
Carbohydrates acid or acid and gas

Microbiology Lab Manual


No Fermentation Acid fermentation Acid Fermenation
with no gas with gas

MATERIALS:
1. selected bacterial species: E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. fermentation test tubes: lactose, sucrose and dextrose - 1 (one) each per person (12
tubes total for a group of 4)

PROCEDURE: Work individually


1. Obtain fermentation media of each carbohydrate type.
2. Label each tube immediately with station # and organism name. The tubes look alike
but contain different sugars.
3. There are 4 organisms. Each person in the group of 4 should choose an organism.
Each person is responsible for inoculating that organism into each of the media types
needed to complete this lab.
4. Using aseptic technique take inoculating loop full of pure culture and inoculated each
of three fermentation test tubes.
5. Place the tube in the appropriate test tube rack for incubation at 37 degrees Celsius for
48 hours. Lids should be loose on the test tubes.
6. Determine which organisms fermented each sugar.
7. Record the results. (Note: acid & gas, acid, or neither).

DIGESTION OF STARCH
Starch is a complex polysaccharide composed of thousands of glucose molecules in straight and
branched chains. Some bacteria make the enzyme amylase which breaks down the
polysaccharide into smaller carbohydrates. The medium used to test for amylase is nutrient agar
that contains starch. Iodine is used to determine whether the organism can digest the starch in
the medium.
amylase
Starch Glucose subunits
Glucose subunits + iodine = clear zone around growth

Microbiology Lab Manual


Clear zone around the streak indicates starch hydrolysis

MATERIALS:
1. selected bacterial species: E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. starch agar plates - 1 plate per person
3. iodine solution

PROCEDURE: Work in groups of 4.


1. Obtain a starch plate.
2. Label the plate with station # and organism name.
3. Inoculate your plate with the same organism you used to inoculate the fermentation
tubes by streaking the loop across the agar plate one time. Do not streak for isolation.
4. Place the plate in the carrier so they can be incubated at 37 degrees Celsius for 24-48
hours.
5. Pour several drops of iodine on the plate over the area where there is growth. Iodine
reacts with the starch to produce a purple blue black complex. However, if the
bacteria have digested the starch, the area around the growth will be clear. DO NOT
confuse the cream colored bacterial growth for the area of starch digestion.
6. Record the results for each organism.

CATALASE TEST

Microbiology Lab Manual


The enzyme catalase breaks down peroxide into water and oxygen. Since some bacteria produce
peroxide during high energy yielding reactions, catalase eliminates this toxic compound in the
bacteria. Catalase also breaks down the peroxide that is used as an antiseptic, therefore, peroxide
is not always a good antiseptic. Anaerobes will be the most susceptible to peroxide.
Catalase
H2O2 H2O + O2 (bubbles)

MATERIALS:
1. selected bacterial species: E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. 3% hydrogen peroxide solution
3. glass slides

PROCEDURES: Work in groups of 4


1. Obtain a small amount of bacteria from the nutrient agar slant with a loop. Use the
same organism you have been using.
2. Place the bacteria on a clean glass slide.
3. Each of the 4 partners should place their slides side by side on the bench top.
4. One person can then add a drop of peroxide to each slide almost simultaneously. The
peroxide should be added directly to the bacteria as quickly as possible.
5. Watch for bubbles produced as the oxygen is released from the breakdown of
peroxide.
6. Watch for bubbles on each of the 4 slides.
7. Record the results.

DNase TEST AGAR

Some bacteria produce the enzyme DNase which breaks long chains of DNA down into
nucleotides. The production of DNase is an identifying characteristic for certain bacteria. The
medium used to test for DNase contains DNA and methyl green. The long chains of DNA bind
the dye. If DNase is produced by an organism and the DNA is broken down, the DNA no longer
binds the dye. In the area of the agar where the DNase has broken down into nucleotides the
agar turns clear. A clear area around the bacterial growth indicates production of DNase. The
color change from green to clear is due to a pH change which occurs when the DNA is broken
down to nucleotides.
DNase
DNA Nucleotide subunit

Microbiology Lab Manual


DNase Positive DNase Negative

MATERIALS:
1. selected bacterial species - E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. DNase test agar - 1 per person

PROCEDURE: Work in groups of 4.


1. Obtain a DNase test agar plate.
2. Label the plate with organism name and your station number.
3. Pick up organisms on the inoculating loop. Use the same organism you have been
using. Your partners will do the other organisms. Streak the loop across the plate
once. Do not streak for isolation.
4. Place the plate in the appropriate carrier for incubation at 37 degrees Celsius for 24-
48 hours.
5. Observe the plates and record the results.

SULFIDE, INDOLE, MOTILITY (SIM) AGAR


SIM medium is used for the differentiation of microorganisms on the basis of hydrogen sulfide
production, indole production and motility.
H2S Production
Some bacteria produce the enzyme cysteine desulfurase which breaks the amino acid cysteine
down into hydrogen sulfide (H2S) gas, pyruvic acid and ammonia. Hydrogen sulfide can be
recognized by its rotten egg smell, but since it is a gas, it dissipates from the tubes. Ferrous
ammonium sulfate in the medium serves as an indicator by combining with the gas, forming an
insoluble black ferrous sulfide precipitate that is seen along the stab and is indicative of H2 S
production.
Cysteine desulfurase
Cysteine H2S + pyruvic acid + NH3

H2S + FeSO4 FeS + H2SO4


(Iron sulfide)
Blackening

Microbiology Lab Manual


Uninoculated
tube

Indole production
The amino acid tryptophan is present in SIM agar as a source of pyruvic acid for organisms. If
tryptophan. Some bacteria produce the enzyme tryptophanase which breaks down the amino
acid tryptophan into indole, pyruvic acid and ammonia. Indole is a product with a putrid smell
that gives fecal material part of its characteristic odor. The pyruvic acid can be utilized in the
Krebs cycle to produce energy for the cell. The ammonia is given off. Indole can be visualized
by the addition of Kovac's reagent (this contains para-amino benzaldehyde which reacts with
indole). If indole is present, a red ring appears at the top of the tube. If no indole is produced,
the reagent sits at the top of the tube and remains an amber color (sometimes the reagent turns
green).
tryptophanase
Tryptophan indole + pyruvic acid + NH3
Indole + Kovacs reagent = Red ring on the surface
Motility
SIM agar may also be used to detect motile organisms. This is a semisolid agar that encourages
the movement of motile organisms away from the line of inoculation. Growth of non-motile
organisms is confined to the line of inoculation.

MATERIALS:
1. selected bacterial species - E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. tubes of SIM medium - 1 per person
3. inoculating needle

PROCEDURE: Work individually


1. Obtain a tube of SIM medium.
2. Label the tube with the organism name and your station number.
3. Using the same organism you have been using, inoculate the tube with a straight wire.
4. Carefully stab all the way to the bottom of the tube without shaking.
5. Place the tube in the appropriate test tube rack for incubation at 37 degrees Celsius for

Microbiology Lab Manual


24-48 hours.
6. Observe the tubes for H2S production, indole and motility.

UREASE TEST

Urea is an end product of protein metabolism in many organisms. The enzyme urease produced
by some bacteria digests urea into ammonia and carbon dioxide. If an organism is grown in
medium containing urea and if the organism makes urease, the urea is broken down as described.
The ammonia produced increases the pH of the medium, which also contains the pH indicator
phenol red. The pH indicator phenol red is affected by the increase in pH. The medium turns a
bright magenta (deep pink) when ammonia is released from urea.

Urease
Urea + H2 O NH3 + CO2 + H2 O

MATERIALS:
1. selected bacterial species - E. coli, S. epidermidis, B. subtilis, P. vulgaris
2. urea slants - 1 per person

PROCEDURE: Work in groups of 4.


1. Obtain a urea slant.
2. Label the tube with the organism name and your station number.
3. Double inoculate this tube with the organism you have been using. With inoculating
needle streak surface of the slant and with same needle stab the deep of the slant.
4. Place the tube in the appropriate test tube rack for incubation at 37 degrees Celsius for
24-48 hours.
5. Observe the results.

Microbiology Lab Manual


RESULTS and OBSERVATIONS

Complete the following table by using A for acid, G for gas, a + for positive and a
- for negative results.

Organism Fermentation Starch DNase Catalase H2S Indole Motility Urease

D S L

Proteus

Bacillus

S. epidermidis

E. coli

Serratia

Legends: D = Dextrose; S = sucrose; L = Lactose

Questions
1. List reasons for studying biochemical characteristics of bacteria.

2. What is the end product of carbohydrate fermentation?

3. What is the name of small test tube in carbohydrate fermentation test tube?

4. What is the name of pH indicator in carbohydrate fermentation test tube?

5. Name the enzyme which breaks down the polysaccharide into smaller carbohydrates?

6. Name the enzyme which breaks down hydrogen peroxide into water and oxygen?

7. Name the enzyme which breaks down DNA into nucleotides?

Microbiology Lab Manual


8. Which amino acids are present in SIM agar?

9. SIM stand for

10. Name the gas which is produced when cysteine is hydrolyzed?

11. What is the end product which causes blackening of the SIM agar?

12. Indole reacts with which agent to produce red ring?

13. Name the enzyme produced by the bacteria which hydrolyzed urea?

14. What is the name of pH indicator in urea agar and what is its function?

Microbiology Lab Manual


Parasitology: Protozoa and Helminths

Since the natural histories of parasitic diseases differ in some important respects from those of
bacterial diseases, they merit a separate session to give you introductory laboratory experience
with parasites, the diseases they cause, and techniques used to diagnose them.

The distinguishing features of parasitic life are the close contact of the parasite with the host in
or on which it lives and its dependency on the host for life itself. This special association has led
to the evolution of three types of adaptations not found in the free-living relatives of the
parasites: loss of competency, special structures, and ecological ingenuity.

Parasites have become so dependent on their hosts for food and habitat that they now experience
a loss of competency to live independently. They usually require a specific host, and many have
lost their sensory and digestive functions; these are no longer important for their survival.

On the other hand, they have developed special structures and functions not possessed by their
free-living relatives that promote survival within the host. One example is special organs of
attachment hooklets and suckers. Parasites also have a tremendously increased reproductive
capacity, which compensates for the uncertainty in finding a new host. Tapeworms, for example,
have fantastically high rates of egg production, reaching up to 100,000 per day.

Ecological ingenuity is demonstrated in the fascinating variety of infecting and transmitting


mechanisms. This has led to very complex life cycles, which contract markedly with the
relatively simple lifestyles of their free-living counterparts. Parasites show quite a range in the
types of life cycles they possess, from species that pass part of each generation in the free-living
state to others that require at least three different hosts to complete the life cycle. Some are
simply transmitted by insects from one human host to the new host, or the insect may act as a
host as well. Many protozoa develop resistant cysts, which enable them to survive in
unfavorable environments until they find a new host. The eggs of flatworms and roundworms
also have a protective coat.

These three strategies promote survival and expansion of the species by providing greater
opportunities for finding and infecting new hosts, which is a continual problem for parasites.
Successful interruption of these cycles to prevent their completion is an important feature of
public health measures used to control diseases caused by parasites.

This exercise is designed to give you some practical experience with representative protozoan
and helminthic parasites and with clinical methods used in their diagnosis and control. Your
study will consist of these procedures:

1. As an introduction, you will have an opportunity to observe the movements and structure
of some living nonparasitic protozoans and worms often found in pond water.

Microbiology Lab Manual


2. Examination of commercially prepared stained blood and fecal slides that contain human
protozoan parasites.
3. Study of the natural history and life cycle of the human parasitic disease schistosomiasis.
This will enable you to see the interaction between stages of the life cycle, environmental
surroundings, and social condition of their human hosts as factors in the epidemiology
and control of the disease.

The following classification of parasites will serve as a guide to the examples you will be
studying in this exercise. It is not a complete listing.

Protozoa

Protozoa, a subkingdom of the kingdom Protista, are unicellular eukaryotic organisms. They
usually reproduce by cell division and are classified mainly according to their means of
locomotion. Only one phyla, the Suctoria, which is closely related to the Ciliophora, does not
contain animal pathogens. The remaining is classified as follows:

Sarcodina

Members of this subphylum move and feed slowly by forming cytoplasmic projections known as
pseudopodia (false feet). They also form both trophozoites (vegetative form) and cysts
(resistant, resting cells). Parasitic members include the amoeba Entamoeba histolytica, which
causes amoebic dysentery. It ingests red blood cells and forms a four-nucleate cyst. It is also
found in animals. Other amoeba species found in humans, such as Entamoeba gingivalis, are
relatively harmless commensals.

Ciliophora

Members of this phylum have many short, hairlike cilia on their body surface that beat
rhythmically by bending to one side. Ciliophora is typified be genera Paramecium. Another
member, Balantidium coli, is a common parasite in swine. It possesses both cyst and trophozoite
form and can infect humans, causing serous results.

Mastigophora

These protozoans propel themselves with one or more long, whip-like flagella. Some have more
than one nucleus, and they usually produce cysts. Different species cause infection in the
intestine, vagina, blood, and tissues. Giardia lamblia causes a mild to severe diarrheal infection.
Trichomonas vaginalis is found in the urogenital region, where it causes a mild vaginitis in
women. Trypanosoma gambiense infects the blood via tsetse fly bites, causing trypanosomiasis,
or African sleeping sickness, in cattle and humans. Cattle and other ungulates serve as a
reservoir for this organism.
Sporozoa

Sporozoa are obligate, non-motile parasites with alternating stages: the sexual reproductive stage
is passed in the definitive insect host, and the asexual phase is passed in the intermediate human

Microbiology Lab Manual


or animal host. The genus Plasmodium includes the malarial species, in which the definitive
host is the female Anopheles mosquito and the intermediate host is humans. Four species in this
genus are P. vivax, P. falciparum, P. ovale and P. malariae. The genus Coccidia includes
important intestinal parasites affecting fowl, cats, dogs, swine, sheep, and cattle. Toxoplasma
gondii is a cat parasite that can harm the human fetus in an infected pregnant woman.

Helminths (Worms)

Helminths are multicellular eukaryotic organisms. Two of the phyla, Platyhelminthes


(flatworms) and Nematoda (roundworms), contain pathogenic worms.

Class Trematoda (Flukes)


Flukes have an unsegmented body, and many have suckers to hold them onto the hosts intestinal
wall. Many flukes have complex life cycles that require aquatic animal hosts. The Schistosoma
species are bisexual trematodes that cause serious human disease. They require polluted water,
snails, and contact with human skin for completion of their life cycles. Clonorchis sinensis and
Fasciola species are liver flukes acquired by eating infected raw fish and contaminated
vegetables, respectively.

Class Cestoda (Tapeworms)


Tapeworms are long, segmented worms with a small head (scolex) equipped with suckers and
often hooklets for attachment to the hosts intestinal wall. The series of segments, or
proglottids, contain the reproductive organs and thousands of eggs. These segments break off
and are eliminated in the feces, leaving the attached scolex to produce more proglottids with
more eggs. The symptoms of Taenia tapeworm infection are usually not serious, causing only
mild intestinal symptoms and loss of nutrition. Not so for the Echinococcus tapeworm, which
causes a serious disease. All tapeworm diseases are transmitted by animals.

Phylum Nematoda

Members of Nematoda (roundworms) occupy an important ecological niche since they are
present in large numbers in very diverse environments, including soil, fresh water, and seawater.
In contrast to the Platyhelminthes, these round, unsegmented worms are coelomate (have a body
cavity), and have a complete digestive tract and separate sexes. This phylum contains many
agents of animal, plant, and human parasitic diseases. Most require only one host and can pass
part of their life cycle as free-living larvae in the soil. Trichinella spiralis requires alternate
vertebrate hosts. Humans become infected when they ingest inadequately cooked meat, such as
pork or bear containing the larval forms in the muscles. Ascaris lumbricoides is probably the
most common worldwide of all the human helminths. Enterobius vermicularis causes pinworm,
a very common condition in children in the United States. Efforts to eradicate it have not been
very successful since pinworm causes little discomfort.
Definitions

Acoelomate. Without a true body cavity. Typical of members of the Phylum Platyhelminthes
(flatworms).

Microbiology Lab Manual


Amoeba. Unicellular organisms with an indefinite changeable form.

Cercaria. The last miracidium stage in which the larvae possess a tail.

Coelomate. With a true body cavity. Typical of members of the Phylum Nematoda
(roundworms).

Commensal. A relationship between two organisms in which one partner benefits from the
association and the other is unaffected.

Cysts. In protozoan morphology, the hard resting stage, dormant, thick-walled.

Definitive host. The host in which the sexual reproduction of a parasite takes place.

Intermediate host. The host that is normally used by a parasite in the course of its life cycle and
in which it multiplies asexually but not sexually.

Merozoites. Schizont nuclei that become surrounded by cytoplasm and bud off as daughter cells
or merozoites.

Miracidium. A free-swimming ciliate larva that seeks out and penetrates a suitable intermediate
snail host, in which it develops into a sporocyst.

Proglottid. Any of the segments of a tapeworm formed in the neck region by a process of
strobilation (transverse fission).

Pseudopodia. Extensions of cytoplasm that aid in engulfing particles and functioning in motility
of amoeboid cells.

Schizont. A stage in the life cycle of Sporozoa in which the nucleus undergoes repeated nuclear
division without corresponding cell divisions.

Scolex. The head of a tapeworm, which is used for attaching to the hosts intestinal wall.

Sporocyst. A stage in the life cycle of certain protozoa in which two or more of the parasites are
enclosed within a common wall.

Trophozoites. The motile feeding stage of protozoa.

Microbiology Lab Manual


Malaria life cycle

The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-
infected female Anopheles mosquito inoculates sporozoites into the human host .
Sporozoites infect liver cells and mature into schizonts , which rupture and release
merozoites . (Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can
persist in the liver and cause relapses by invading the bloodstream weeks, or even years
later.) After this initial replication in the liver (exo-erythrocytic schizogony ), the
parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony
). Merozoites infect red blood cells . The ring stage trophozoites mature into schizonts,
which rupture releasing merozoites . Some parasites differentiate into sexual
erythrocytic stages (gametocytes) . Blood stage parasites are responsible for the
clinical manifestations of the disease.
The gametocytes, male (microgametocytes) and female (macrogametocytes), are
ingested by an Anopheles mosquito during a blood meal . The parasites multiplication
in the mosquito is known as the sporogonic cycle . While in the mosquito's stomach,
the microgametes penetrate the macrogametes generating zygotes . The zygotes in
turn become motile and elongated (ookinetes) which invade the midgut wall of the
mosquito where they develop into oocysts . The oocysts grow, rupture, and release
sporozoites , which make their way to the mosquito's salivary glands. Inoculation of
the sporozoites into a new human host perpetuates the malaria life cycle.

Microbiology Lab Manual


Toxoplasma gondii
life cycle

The only known definitive hosts for Toxoplasma gondii are members of family Felidae (domestic cats
and their relatives). Unsporulated oocysts are shed in the cats feces . Although oocysts are usually
only shed for 1-2 weeks, large numbers may be shed. Oocysts take 1-5 days to sporulate in the
environment and become infective. Intermediate hosts in nature (including birds and rodents) become
infected after ingesting soil, water or plant material contaminated with oocysts . Oocysts transform
into tachyzoites shortly after ingestion. These tachyzoites localize in neural and muscle tissue and
develop into tissue cyst bradyzoites . Cats become infected after consuming intermediate hosts
harboring tissue cysts . Cats may also become infected directly by ingestion of sporulated oocysts.
Animals bred for human consumption and wild game may also become infected with tissue cysts after
ingestion of sporulated oocysts in the environment . Humans can become infected by any of several
routes:

eating undercooked meat of animals harboring tissue cysts .


consuming food or water contaminated with cat feces or by contaminated environmental
samples (such as fecal-contaminated soil or changing the litter box of a pet cat) .
blood transfusion or organ transplantation .
transplacentally from mother to fetus .

In the human host, the parasites form tissue cysts, most commonly in skeletal muscle, myocardium,
brain, and eyes; these cysts may remain throughout the life of the host. Diagnosis is usually achieved by
serology, although tissue cysts may be observed in stained biopsy specimens . Diagnosis of congenital
infections can be achieved by detecting T. gondii DNA in amniotic fluid using molecular methods such as
PCR .

Microbiology Lab Manual


Tapeworm Life cycle

Life cycle of Taenia spp.


Taeniasis is the infection of humans with the adult tapeworm of Taenia saginata, T. solium
or T. asiatica. Humans are the only definitive hosts for these three species. Eggs or gravid
proglottids are passed with feces ; the eggs can survive for days to months in the
environment. Cattle (T. saginata) and pigs (T. solium and T. asiatica) become infected by
ingesting vegetation contaminated with eggs or gravid proglottids . In the animal's
intestine, the oncospheres hatch , invade the intestinal wall, and migrate to the striated
muscles, where they develop into cysticerci. A cysticercus can survive for several years in
the animal. Humans become infected by ingesting raw or undercooked infected meat . In
the human intestine, the cysticercus develops over 2 months into an adult tapeworm, which
can survive for years. The adult tapeworms attach to the small intestine by their scolex
and reside in the small intestine . Length of adult worms is usually 5 m or less for T.
saginata (however it may reach up to 25 m) and 2 to 7 m for T. solium. The adults produce
proglottids which mature, become gravid, detach from the tapeworm, and migrate to the
anus or are passed in the stool (approximately 6 per day). T. saginata adults usually have
1,000 to 2,000 proglottids, while T. solium adults have an average of 1,000 proglottids. The
eggs contained in the gravid proglottids are released after the proglottids are passed with
the feces. T. saginata may produce up to 100,000 and T. solium may produce 50,000 eggs
per proglottid respectively.

Microbiology Lab Manual


MICROBIOLOGICAL MEDIA

Microorganisms require certain basic nutrients and physical factors for the sustenance of life.
However, their specific requirements vary greatly. Understanding these needs is essential for the
successful growth of microorganisms in the laboratory.

Special purpose media are available for the functions such as:

SELECTIVE MEDIA
A selective medium is used to select specific group of bacteria. A selective medium contains
chemical substances that inhibit the growth of one type of bacteria while allowing the growth of
another type of bacteria, thus facilitating bacterial isolation.

DEFFERENTIAL MEDIA
A differential medium can distinguish between morphologically and biochemically related
groups of bacteria. A differential medium contains chemical compounds that following
inoculation produces a characteristic change in the appearance of bacterial growth and /or the
medium surrounding the colonies, which permits differentiation.

Sometimes selective and differential characteristics are combined in a single medium.

ENRICHED MEDIA
An enriched medium has been supplemented with highly nutritious material, such as blood,
serum or yeast extract, for the purpose of growing fastidious bacteria.

Microbiology Lab Manual


Table of Microbiological Media Characteristics

Medium Selective Differential Selective Differential Additional Reaction and Description


agent agent components
Mannitol Salt Mannitol-fermenter
Agar (MSA) yes yes 7.5% Mannitol Phenol red Positive media turns yellow
(Used for NaCl (pH around the colonies.
halophiles) indicator) None-mannitol fermenter
Negative no media color
change.
Blood Agar Alpha Hemolysis- (partial)
(used to (Colony surrounded by
determine No Yes None Based on None khaki green halo)
hemolytic (based on hemolysis Beta Hemolysis-(complete)
patterns) hemolysis) pattern (Colony surrounded by clear
halo)
Gamma Hemolysis- (None)
(No halo, no hemolysis)

Mitis-Salivarius Colony Characteristics:


Agar Strep. Mitis-tiny pinpoint
(Used for Yes No Tellurite None None dark blue colonies
Viridans Strep. Salivarius- blue gum
Streptococcus) drop shape colonies

Chocolate Agar All Neisseria produce


(nutrient oxidase. Oxidase reagent
enriched to No No None None None (tetramethylparaphenyalene-
promote growth diamine hydrochloride)
of Neisseria) added acts as an artificial
electron acceptor instead of
oxygen. If oxidase is present
colonies turn blue-black

MacConkey Bile salts Fermenters: Pink to Brick


Agar Yes Yes and Lactose Neutral red red pigmented colonies. E.
(used for Crystal coli
Enteric) violet Non-fermenters: Colorless,
clear, cream colonies.
Proteus

Eosin- Eosin & Lactose Fermenters: Dark


Methylene Blue Yes Yes Methylen Eosin & pigmented colonies
Agar (EMB) e Blue Methylene Non-fermenters: Colorless,
(Used for Blue clear or cream
Enteric) E. coli: Dark pigmented
colonies with green
metallic sheen

Microbiology Lab Manual


THE STAPHYLOCOCCI

OBJECTIVES:
1. Differentiate between Staphylococcus aureus and Staphylococcus epidermidis colony
characteristics
2. Differentiate pathogenic Staphylococci from non-pathogenic Staphylococci by
biochemical tests.
3. Differentiate Staphylococci from Streptococci.
4. Discuss different tests performed to differentiate between Staphylococcus aureus and
Staphylococcus epidermidis.
5. Interpret data from the biochemical tests needed for diagnosis of Staphylococci.
5. Learn the name of the diseases caused by Staphylococcus aureus and Staphylococcus
epidermidis.

The Staphylococci are Gram (+) cocci occur in clusters, inhabits human skin and nose. Two
representative organisms from this group are S. aureus and S. epidermidis. The colonies of S.
aureus are round and golden yellow in color. The colonies of S. epidermidis are white color
small pinpoint colonies. They are salt tolerant (NaCl to 10%).

S. aureus causes food poisoning, acute bacterial endocarditis, toxic shock syndrome, boils,
wound infections, osteomyelitis and meningitis. S. epidermidis is a normal flora of the skin. It is
not usually involved in disease under normal conditions. Under certain conditions, S.
epidermidis can be involved in sub-acute endocarditis, catheter infections and prosthetic joint
infections.

Mannitol Salt Agar (MSA)


Mannitol Salt Agar is used for isolation and differentiation of pathogenic staphylococci,
especially S. aureus. Mannitol Salt agar contains the carbohydrate mannitol, 7.5% sodium
chloride (NaC1) and pH indicator phenol red. Phenol red will turn yellow below pH 6.8.
In this exercise, Staphylococci will be isolated from the body. Since the organisms are salt
tolerant (halophiles), sodium chloride makes this medium selective for staphylococci. The
pathogenic species of Staphylococcus ferments mannitol and produce acid, which turns the pH
indicator yellow. Nonpathogenic staphylococcal species grow, but produce no color change. S.
aureus can be differentiated from S. epidermidis on the basis of mannitol fermentation. S.
aureus ferments mannitol whereas S. epidermidis does not.

Coagulase Test
The pathogenic Staphylococci produce coagulase. S. aureus makes coagulase and is a pathogen.
S. epidermidis does not produce coagulase and is not considered a pathogen. Plasma clots in the
presence of coagulase. If plasma is inoculated with an organism and a clot forms, then the
organism is producing coagulase and, it is a pathogen.

The two tests for pathogenic Staphylococci are the fermentation of mannitol and the production
of coagulase. Both S. epidermidis and S. aureus are catalase (+)

Microbiology Lab Manual


MANNITOL - SALT AGAR INOCULATION

MATERIALS:
1. cotton swabs
2. sterile saline
3. Mannitol-salt agar plates (2 per student)
4. cultures of S. aureus and S. epidermidis

PROCEDURE: Work in groups of 2.


1. Make a control four quadrant streak plate using pure culture of either S. aureus or
S. epidermidis on mannitol salt agar
2. Each student will take two Mannitol salt agar, label it with your initials, station
number and write nose on first plate and skin on second plate.
3. Use cotton tip applicator take specimen from nose and make first quadrant on
plate labeled as nose and then make the remaining three quadrant streaks using
inoculation loop.
4. Next use another fresh cotton tip applicator, wet it with distilled water.
5. Rub a moist skin surface with the cotton swab.
6. Make first quadrant on the plate labeled as skin and then make the remaining
three quadrant streaks using inoculation loop.
8. Place the all the plates for your group in the appropriate carrier for incubation at
37 degrees Celsius for 48 hours.
9. Observe the plates for fermentation of mannitol.

COAGULASE PLASMA TEST

MATERIALS:
1. tubes containing 0.5ml coagulase plasma (one per group of two students).
2. cultures of S. aureus and S. epidermidis
3. Pasteur pipettes and bulbs

PROCEDURE: Work in pairs.


1. Inoculate a tube of coagulase plasma with either S. aureus or S. epidermidis. Your
partner will do the other. Leave the caps loose. Label tubes with the organism
name and your station number.
2. Place the tubes in the appropriate rack for incubation overnight at 370 C.
3. Observe the tubes for coagulation.

Microbiology Lab Manual


CATALASE TEST

MATERIALS:
1. Culture plates
2. 3% hydrogen peroxide

PROCEDURE:
1. Add two to three drops of hydrogen peroxide in each plate

RESULTS and OBSERVATIONS

Media and Enzyme Reactions

Organism Mannitol Coagulase Catalase


fermentation

Staphylococcus aureus

Staphylococcus
epidermidis

Mannitol fermentation: positive or negative


Catalase: positive or negative,
Coagulase: positive or negative

Questions

1. Describe genus Staphylococci?

2. Name two representative organisms in this genus.

3. Describe mannitol salt agar.

4. What is the name of extracellular protein secreted by Staphylococcus aureus?

Microbiology Lab Manual


THE STREPTOCOCCI

OBJECTIVES:
1. Isolate Streptococci from various parts of the upper respiratory tract.
2. Recognize and interpret the different types of hemolysis (alpha, beta, and gamma).
3. Discuss diseases caused by genus Streptococci.
4. Discuss the Streptococcus viridans and their relation to disease of the oral cavity.
5. Use the biochemical tests necessary to select and identify Streptococci.
6. Discuss and use appropriate Universal Precautions for Blood Borne Pathogens.

The Streptococci are a group of Gram (+) many are facultative anaerobic cocci that form chains.
The organisms grow best on enriched media (e.g. blood agar) in an atmosphere of 5-10% CO2.
Many of the streptococci are normal flora of the skin, upper respiratory tract, mouth and the
intestines. The pathogen in this S. pyogenes causes strep throat, necrotizing fasciitis, scarlet
fever, rheumatic fever, impetigo, erysipelas, toxic shock syndrome and glomerulonephritis. The
streptococci are classified by the hemolytic reaction on blood agar plates. The organisms can be
partially classified on the basis of hemolysis.
() Alpha-hemolysis
Organisms are alpha-hemolytic if the red blood cells are incompletely destroyed and produce
green zone around the colony. S. mitis and S. pneumoniae are alpha-hemolytic.
() Beta- hemolysis
Organisms are beta-hemolytic if the blood cells in the agar are completely destroyed by the
hemolysin produced by the bacteria. A clearing is evident around the colonies of beta-hemolytic
organisms. S. pyogenes and S. agalactiae is beta-hemolytic.
() Gamma- hemolysis
The organisms that do not hemolyze red blood cells at all are gamma-hemolytic. S. lactis and S.
faecalis are gamma-hemolytic.

In this exercise, organisms from the upper respiratory tract will be examined. Normal flora of the
upper respiratory tract include - hemolytic Streptococci, Neisseria speciesStaphylococcus
aureus and other Staphylococcus species, - hemolytic Streptococci, and - hemolytic
Streptococci (in a few people). A large number of -hemolytic Streptococci indicate infection or
carrier state.

Some of the streptococci found in the oral cavity contribute to tooth decay. The viridans group
of streptococci such as S. mutans, S. mitis and S. salivarius produce acid which attacks tooth
enamel. Mitis-salivarius medium can be used to grow these organisms. The medium contains
tellurite which selects for the viridans group of streptococci. The colonies will be blue. This
medium is not differential. S. mitis produces pinpoint colonies while S. salivarius produces
gumdrop shaped colonies.

Microbiology Lab Manual


STREPTOCOCCI FROM THE UPPER RESPIRATORY TRACT (Oropharynx)

MATERIALS:
1. tongue depressors
2. sterile swabs
3. blood agar plates - one (1) per person.
4. protective gown, goggles, gloves, and mask obtained from an instructor.

PROCEDURE: Work in groups of 2.


1. Obtain throat swabs from each other. Use a tongue depressor to hold the tongue down. Rub the
back of the oropharynx with the cotton swab at the level of the uvula. Try not to make the
person gag. Follow the OSHA guidelines for handling saliva, a body fluid. The person
performing the throat swab must be wearing a gown, goggles, gloves and mask.
2. Streak the first section of a blood agar plate with the cotton swab. Continue streaking the plate
for isolation with an inoculating loop. Label the plates with throat and your station number.
3. Place the plates in the appropriate carrier for incubation at 37 degrees Celsius for 24-48 hours.
4. Record the results.

STREPTOCOCCI OF THE ORAL CAVITY

MATERIALS:
1. cotton swabs
2. Mitis-Salivarius medium (one plate per person)

PROCEDURE: Work in groups of 2.


1. Obtain a sample from your own gingival crevice with a cotton swab. Swab where the tooth and
the gum line meet.
2. Streak the first section of a mitis-salivarius plate with the cotton swab.
3. Continue streaking for isolation with an inoculating loop.
4. Place the plates in the appropriate carrier for incubation at 37 degrees Celsius for 48 hours.
5. Observe the plates for dark blue colonies.
6. Record the results.

Microbiology Lab Manual


RESULTS and OBERVATIONS

Blood Agar

Organism Blood agar hemolysis

Streptococcus pneumoniae

Streptococcus pyogenes

Hemolysis: Alpha hemolysis, Beta hemolysis or Gamma hemolysis

Mitis- Salivarius agar

Organism Mitis-Salivarius medium colonies

Streptococcus mitis

Streptococcus salivarius

Mitis-Salivarius medium: color and characteristics of the colonies

Questions
1. Describe genus Streptococcus.

2. Describe blood agar.

3. Describe different types of hemolysis and name the species which cause different types of hemolysis.

4. According to OSHA guideline what should be used while working with body fluids?

5. Describe Mitis-salivarius agar

6. Describe colonies of Streptococcus mitis and Streptococcus salivarius on Mitis-salivarius agar.

Microbiology Lab Manual


CCUPATIONAL EXPOSURE TO BLOODBORNE PATHOGENS

Occupational Safety and Health Administration (OSHA)

Bloodborne Pathogens means pathogenic organisms that are present in human blood and can cause disease in
humans. These pathogens include but are not limited to hepatitis B virus (HBV) and human immunodeficiency
virus (HIV).
Other Potentially Infectious Materials means the following body fluids: semen, vaginal secretions, cerebrospinal
fluid, synovial fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, any body fluid visibly
contaminated with blood and ALL body fluids in situations where it is difficult to differentiate between body
fluids.
Universal Precautions: According to the concept of Universal Precautions, all human blood and certain human
body fluids are treated as if known to be infectious for HIV, HBV, and other bloodborne pathogens.

BODY SUBSTANCE ISOLATION:


Body substance isolation refers to the idea that containment of body fluids via aseptic practices is at least
important or more so than Universal Precautions.
Methods of Compliance: The following precautions shall be observed to prevent contact with blood and other
potentially infectious materials.

1. Work in designated area ONLY.


2. Wipe area down with a bleach solution.
3A. Handling urine:
WORKING ALONE-
a. Obtain a urine sample and return to the designated area.
b. Test the urine sample.
c. Wipe up any spills immediately and place the contaminated toweling in the biohazard bag.
Disinfect the area and materials with bleach.
d. Pour the urine into the sewer system.
e. Place the sample cup in the biohazard bag.

WORKING IN GROUPS-
a. All members of the group must wear lab coats and gloves. Goggles and masks are available.
b. Obtain and test a urine sample according to the procedure in the lab manual.
c. Wipe up any spills immediately and place the contaminated toweling in the biohazard bag.
Disinfect the area and materials with bleach. Dispose soiled gloves into the biohazard bag.
Wash hands and re-glove with sterile gloves.

3B. Handling blood:


WORKING ALONE-
a. Follow the procedure for obtaining blood as outlined in the manual.
b. Immediately after use, contaminated sharps shall be placed in puncture resistant biohazard
containers for decontamination. Contaminated sharps includes lancets, slides and glassware.
c. Non-sharp material contaminated with blood will be placed in appropriate biohazard bags.
d. Perform the blood test.
e. Wash hands.

WORKING IN GROUPS-
a. All members of the group, not giving blood, will wear lab coats, gloves, masks and goggles.
b. Students will work ONLY with their own blood.

Microbiology Lab Manual


c. Follow the procedure for obtaining blood as outlined in the manual.
d. Immediately after use, contaminated sharps shall be placed in a puncture resistant biohazard
container for decontamination. Contaminated sharps includes lancets, slides and glassware.
e. Non-sharp material contaminated with blood will be placed in biohazard bags.
f. Perform the blood test.
g. Remove contaminated mask and gloves and place them in the biohazard bag. Remove goggles.
h. Wash hands.

3C. Throat cultures: WORK IN PAIRS


1. The person taking the throat culture must wear gloves, lab coat, mask and goggles.
2. Take the throat culture using a sterile cotton swab and a tongue depressor using aseptic
technique.
3. Streak a blood agar plate with the cotton swab.
4. Once the swab and tongue depressor are placed in the biohazard bag, remove the gloves and
mask. Place the gloves and mask into the biohazard bag.
5. Remove goggles.
6. Continue to wear the lab coat.

3D. Collecting saliva: WORK ALONE


1. Chew paraffin or gum placed in the mouth using aseptic technique according to the procedure
described in the lab manual.
2. Spit carefully into the cup given to you.
3. Using aseptic technique transfer some saliva, with a loop, to an agar plate.
4. Once the test is completed the area is to be wiped down with a bleach solution.
5. Hands should be washed immediately or as soon as feasible after removal of gloves, masks,
goggles or lab coats.
6. Gloves contaminated with blood or body fluids should be removed immediately and placed in
the biohazard bag. Hands should be washed and regloved with sterile gloves.
7. Goggles contaminated with blood or body fluids should be removed before removing gloves and
placed in the appropriate container for decontamination.
8. All procedures involving blood or other potentially infectious materials shall be performed in
such a manner as t minimize splashing, spraying, splattering and generating droplets of these
substances. If any of these should occur, notify the instructor immediately so that the clean up
can be supervised.
9. Exposure to blood or body fluids will be reported to the lab instructor for documentation.
10. Eating, drinking, smoking, applying cosmetics or lip balm and handling contact lenses are
PROHIBITED in work areas where there is reasonable likelihood of contamination.
11. Any cuts, inflammation of scrapes on the skin will be covered with a sterile bandage.
12. Pregnant students, students taking immunosuppressive drugs or students with any medical
condition which might necessitate special precautions in the laboratory should inform the
instructor and obtain a waiver signed by a physician.

_______________________________
Print Name:

_______________________________ ___________________
Signature Date

Microbiology Lab Manual


THE NEISSERIA

OBJECTIVES:

1. Isolate genus Neisseria from the upper respiratory tract (URT).


2. Perform the tests needed to identify Neisseria from other flora of the URT.
3. Describe chocolate agar medium.

Some of the Neisseria such as N. sicca, N. mucosa and N. subflava are normal flora of the upper respiratory
tract. Therefore, Gram (-) diplococci in the oropharynx can be normal. The two pathogens in this group are N.
meningitidis and N. gonorrhoeae which cause meningitis and gonorrhea respectively. Ten to thirty percent of
healthy asymptomatic individuals are carriers of N. meningitidis. These organisms require enriched media to
grow and prefer a 5-10% CO2 atmosphere. The Neisseria produce oxidase. The production of oxidase is
observed when tetramethylparaphenylene-diamine hydrochloride (oxidase reagent) is added directly to colonies
on a chocolate agar plate. This dye is an artificial electron acceptor instead of oxygen. The reagent is colorless
when reduced and when oxidized it turns purple and eventually black.

A non-selective, enriched growth medium used to grow genus Neisseria is called chocolate agar. This medium
contains hemolyzed sheep red blood cells and agar. Hemolyzed RBC's leak out extra nutrients. Because this
medium is so enriched almost any organism will grow on it, and this medium is not considered selective. There
is no characteristic of the medium to make it differential either.

Thayer-Martin Selective Agar is a selective medium for culturing and primarily isolating pathogenic Neisseria
gonorrhoeae and Neisseria meningitidis from specimens. It contains Chocolate II Agar with vancomycin,
colistin and nystatin, it is formulated to minimize the overgrowth of gonococci and meningococci by
contaminants, to suppress the growth of saprophytic Neisseria species and to enhance the growth of pathogenic
Neisseria.
Typical colonial morphology on Thayer-Martin Selective Agar is as follows:
Neisseria gonorrhoeae ..................Small, grayish-white to colorless, mucoid
Neisseria meningitidis ..................Medium to large, blue-gray, mucoid

MATERIALS:
1. cotton swab
2. chocolate agar plate - neither selective or differential - (one per person)
3. tongue depressor

PROCEDURE: Work in groups of 2.


1. Obtain a throat swab from each other. Use a tongue depressor to hold the tongue down. Rub the
back of the oropharynx with the cotton swab behind the uvula. Don't gag your partner. Follow
the OSHA safety guidelines as for the Strep. lab.
2. Streak the first section of a chocolate agar plate with the cotton swab. Continue streaking for
isolation with an inoculating loop. Label the plate with throat and your station number.
3. Place the plates in the appropriate carrier for incubation at 37 degrees Celsius for 48 hours.
4. Look at the colonies. Record the results.
5. Perform the oxidase test. Add a small amount of oxidase reagent directly to a few colonies.
Note any colonies that turn blue-black.
6. Record the results.
7. Do a Gram stain on the blue-black colonies.

Microbiology Lab Manual


RESULTS and OBSERVATIONS

Oxidase Test Reaction

Microorganism Oxidase positive or negative

Neisseria

Questions
1. Describe genus Neisseria

2. Describe chocolate agar.

3. Name the species of genus Neisseria present in upper respiratory tract as normal flora.

4. Name the bacteria and diseases caused by genus Neisseria.


a.
b.

5. Which enzyme is secreted by the genus Neisseria?

Microbiology Lab Manual


THE ENTERIC BACTERIA

OBJECTIVES:

1. Discus Enterobacteriaceae family and where these bacteria are found.


3. Define Gram negative enteric rods.
2. Perform and interpret the biochemical tests needed to differentiate selected Enterics.
3. Define the difference between selective and differential media.
4. Name of some of the enteric bacteria and diseases caused by them.

The Gram (-) non-sporeforming rods are called the enteric bacteria. The majority of them are normal flora of
the gastrointestinal tract. They belong to the family Enterobacteriaceae. The major pathogens are Salmonella
typhi causes typhoid fever, Shigella causes shigellosis or bacillary dysentery, Escherichia coli cause travelers
diarrhea, UTI, diarrhea in infants and Proteus causes UTI.

Isolation of organisms from the GI tract can be difficult because the gut has a wide variety of organisms. The
enteric bacteria can be isolated on media that are both selective and differential.

MacConkey Agar
It is a selective medium, selective agent is bile salt and crystal violet inhibits Gram-positive cocci and allow
Gram-negative organism to grow. It is also a differential medium, differential agent is lactose. Lactose
fermentation causes drop in pH around the colony and color change in the pH indicator neutral red. Lactose
fermenting organisms take up the dyes and form pigmented (pink to brick red) colonies. The colonies of the
non-fermenters are colorless.

Eosin-methylene Blue Agar


It is a selective medium, selective agent is eosin and methylene blue. It is also a differential medium,
differential agent is lactose. The two dyes inhibit Gram (+) organisms. Lactose fermenters will form dark
pigmented colonies while the non-fermenters will form colorless colonies. E. coli produces colonies with a
black center and a metallic green sheen caused by large quantity of acid that is produced and that precipitate the
dyes onto the growths surface.

Triple Sugar Iron Agar


Further differentiation of the enterics can be done with triple sugar iron agar (TSIA). This medium contains the
three sugars glucose- 0.1%, lactose- 1.0% and sucrose 1.0% as well as the pH indicator phenol red, iron ions,
cysteine amino acid and peptides. When the carbohydrates are fermented, acid production is detected by change
in the color. The slants are inoculated on the surface with a loop as well as the deep portion by stabbing with a

Microbiology Lab Manual


wire. This one medium can determine fermentation of the three sugars, acid and CO2 gas production and H2S
production. If glucose is fermented, the agar turns yellow at first. Then the slant reverts to red while the deep
remains yellow. (This is reported as red over yellow). If lactose or sucrose is fermented the agar turns
completely yellow and stays that way. (Reported yellow over yellow). If none of the sugars are fermented the
agar turns a red alkaline color (reported as red over red). Gas production can be detected by cracks, splits and
bubbles in the agar. If H2S is produced an interaction between the H2S and the iron ions produces FeS (black
precipitate) in the agar.

IMViC Test
A useful series of four tests called the IMViC Test is used to further identify the enterics. The "I" stands for
indole. The "M" stands for methyl red. The "V" stands for Vogues-Proskauer. The "i" aids in pronunciation.
The "C" stands for citrate. The indole test is done as previously described in the biochemistry experiment.

Indole production
In indole broth amino acid tryptophan is present. Some bacteria produce the enzyme tryptophanase which
breaks down the amino acid tryptophan into indole, pyruvic acid and ammonia. Indole is a product with a
putrid smell that gives fecal material part of its characteristic odor. The pyruvic acid can be utilized in the
Krebs cycle to produce energy for the cell. The ammonia is given off. Indole can be visualized by the addition
of Kovac's reagent (this contains para-amino benzaldehyde which reacts with indole). If indole is present, a red
ring appears at the top of the tube. If no indole is produced, the reagent sits at the top of the tube and remains
an amber color (sometimes the reagent turns green).

Methyl Red-Voges-Proskauer (MR-VP) medium


The methyl red test depends on the organism's ability to ferment glucose. All enterics initially produce pyruvic
acid from glucose metabolism. Some enterics subsequently use the mixed acid pathway to metabolize pyruvic
acid to other acids, such as lactic acid, acetic acid, and formic acids. If glucose is fermented, the acid produced
will cause the methyl red (a pH indicator) to be red. Methyl red is yellow in alkaline conditions. NOTE: This is
the reverse of phenol red. The Vogues-Proskauer test looks for the production of acetylmethylcarbinol from
glucose. Acetylmethylcarbinol causes a red color to appear when fifteen drops of alpha naphthol (VPA1) and
five drops of potassium hydroxide (VPB2) are added to the tube of MR-VP medium that has been inoculated
and incubated. A lack of color change after the reagents are added indicates a negative VP test.

MR test
Glucose pyruvic acid

Pyruvic acid lactic acid, acetic acid and formic acid (mixed acids)

Mixed acids + methyl red reagent = red color

VP test
Glucose pyruvic acid

Pyruvic acid acetylmethylcarbinol

Acetylmethylcarbinol + alpha-naphthol + KOH = red color

Microbiology Lab Manual


Simmons Citrate Test
This test looks at the ability of an organism to use citrate as a sole carbon source for energy production. When
citrate is utilized alkaline by-products are formed, and the pH of the medium goes up. Citrate medium contains
the pH indicator bromothymol blue which is green at neutral pH and blue at alkaline pH. If the organism uses
citrate as its carbon source, the medium turns deep blue.

ISOLATION OF ENTERICS

MATERIALS:
1. MacConkey agar three plates
2. EMB agar three plates
3. Pure culture of E. coli, Proteus and Enterobacter aerogenes.

PROCEDURE: Work as a group.


1. Label your plates with microorganism and your station number.
2. Using a loop, make four quadrant streak plate using E. coli, Proteus and Enterobacter aerogenes
on each MacConkey agar plate and on EMB plates for isolation of colonies.
3. Place the plates in the appropriate carrier for incubation at 37 degrees Celsius for 48 hours.
4. Observe the colonies for evidence of growth, lactose fermentation and any other characteristics.
5. Record the results.
6. Do a Gram stain on an isolated colony.

TRIPLE SUGAR IRON AGAR INOCULATION

MATERIALS:
1. Pure cultures of E. coli, Proteus and Enterobacter aerogenes.
2. TSIA (one tube per person)

PROCEDURE: Work in a group.


1. Label your tube with your station number and microorganism.
2. Use pure culture of either E. coli or Proteus.
3. Using inoculating needle streak slant and stab butt.
4. Keep the cap loose.
5. Place the TSIA tubes in the appropriate test tube rack for incubation at 37 degrees Celsius for 48
hours.
6. Observe the tubes or acid, gas and/or H2S production.

Microbiology Lab Manual


IMViC TEST
MATERIALS:
1. Pure cultures of E. coli, Proteus and Enterobacter aerogenes.
2. Indole broths for the indole test
3. MR-VP medium
4. Simmons Citrate agar

PROCEDURE: Work in a group.


1. Label your tubes with the name of the organism and your station number.
2. Inoculate 1 indole broth, 2 MR-VP broths and 1 citrate agar with either E. coli, Proteus or
Enterobacter.
3. Inoculate the indole broth and the MR-VP broths using a loop.
3. Inoculate the citrate tube twice. Using an inoculating needle streak the slant and stab deep.
4. Keep the caps loose.
5. Place the tubes in the appropriate test tube rack for incubation at 37 degrees Celsius for 48 hours.
6. Complete the IMViC test as follows:
a. Add Kovac's reagent (5-8 drops) to the indole broth to determine if indole was produced.
Look for a red color.
b. Add one drop of methyl red to one MR-VP broth. The other group will add methyl red to
one of their MR-VP broths. Look for a red color.
c. Add 15 drops of VPA reagent and 5 drops of VPB reagent in each test tube.
d. Keep the tube in water bath.
e. Look for change in color. If it turns red then the test is positive.
e. Look at the citrate agar tubes for a blue color.
f. Record the result.

Microbiology Lab Manual


RESULTS and OBERVATIONS
Selective and Differential Media Reaction color and colony characteristics

Microorganism MacConkey Agar Eosin-methylene blue agar

E. coli

Proteus
Enterobacter aerogenes

Triple Sugar Iron Agar Test Reactions

Microorganism Slant color Butt color Sugar Gas production H2S production
fermentation
E. coli

Proteus

Enterobacter
aerogenes
Sugar fermentation: red/yellow = Glucose, yellow/yellow = Lactose and or Sucrose and red/red = no
reaction

IMViC Reactions

Microorganism I MR VP C

E. coli

Proteus

Enterobacter
aerogenes

Legends: I = Indole; MR = Methyl red; VP = Voges-Proskauer; C= Simmons citrate agar

Microbiology Lab Manual


Questions
1. Define enteric bacteria.

2. Describe:
MacConkey agar

Eosin-methylene blue agar.

3. List constituents present in Triple Sugar Iron Agar (TSIA).

4. In TSIA agar, red/yellow indicates which sugar fermentation?

5. In TSIA agar, yellow/yellow indicates which sugar fermentation?

6. In IMViC series
a. I stands for
b. M stands for
c. Vi stands for
d. C stands for

7. Why is it unlikely that an organism will be positive for both the methyl red and Voges-Proskauer tests?

8. Name the enteric bacteria and diseases caused by them.


a.
b.
c.
d.

Microbiology Lab Manual


Instructions for Completing Your Unknown

1. Your unknown is worth 30 points when completed in full. If you do not complete all of your
unknown work, or if you are incorrect on some of it, you may receive partial points, as described
below.

2. You will work in a group of 4 students


Record your names, unknown number and all results Legibly on your worksheet.

3. You will be assigned an unknown.


Make a four quadrant streak plate with your unknown and incubate it for isolated colonies.
(Plate will be graded based on growth in four quadrants and isolated colonies in the third and
fourth quadrant.)
Observe and record colony characteristics shape, margins, pigment etc.
Perform a Gram Stain isolated colony from an isolated colony from your four quadrant streak
plate. Make two slides for safety, perform a Gram Stain on both and record the morphology,
arrangement, and Gram reaction on your work sheet.

4. Your instructor must observe your gram stain to receive credit. If you are correct in form,
morphology and arrangement, you may continue to the next step. If you are not, you will be given a
second chance (2 points will be deducted). You may need to make another gram stain, or just
reevaluate what you see. Dont just guess!

5. When you have determined morphology, arrangement and gram reaction correctly, you will choose
appropriate biochemical tests.
Choose appropriate biochemical media relevant to solve your unknown organism.
Label all media with your station number and your known number.
Inoculate biochemical tests and incubate them.
You may use your lab book and textbook as references. Do not copy material from lab notes word
for word.
Read biochemical reactions and record results.
Using the biochemical evidence and the information determined from observing the growth,
morphology, and gram reaction determine the genus and species of your unknown.

6. Show your instructor your results. If you are correct, your paper will be signed off. If you are
incorrect, you will be given a second chance. Think, do not guess.

Microbiology Lab Manual


Microbiology 5205 Unknown Lab Report (5% of your grade)

Writing the report (18 points)

I. Introduction
This should include the identified genus and species. Describe its habitat, mode of transmission, growth
requirements and name of diseases found in humans. The report should include if appropriate issues
the organism can cause in the environment that can also impact human health.

II. Material and Methods


Materials: Every day in lab, keep a running list of all materials, media, reagent, instruments that you use.
These should be presented in a table format in the report.

Methods: Without copying from on-line lab manual. List all tests that were performed and the relevance
of each test in determining the name of the unknown specimen. Do Not list step by step instructions on
how to perform the tests (i.e. Gram stain was performed to determine .; Do not write out how to
perform each section of a gram stain).

III. Results
Present your data in table format listing the tests performed, your observations and the results. For example:
Gram Stain Pink rods Gram (-) bacillus

Also create is a dichotomous key (division into two parts) formatting the correct order of tests and the
identification of the organism.

IV. Discussion and Conclusion


In this section you will discuss your results and any problems or questions you encountered along the way.
Discuss your inclusion/exclusion for the tests you performed and the results you obtained in determining your
bacterial genus and species.

Your conclusion should include application of the techniques used to real world situations that relate to your
identified organism. Your discussion and conclusion should be reported in a concise manner, and there must
be correlation to what is presented in the introduction.

V. Format
You report must be typed and printed with a cover sheet including the name of the organism identified and all
of the names of those in your lab group. The worksheet must be attached to the end of the report.

The report itself should be two to four pages double spaced, 12 size font, spellings and grammar counts. In
your report include all headings. If you include pictures you have taken of slides in the report it references
the slide for the color then the report must be submitted in color. If you will not follow the format points
will be deducted.

VI. References
Your report needs to include two references in addition to the textbook and/or lab manual. Wikipedia is Not
an acceptable reference. If you use Wikipedia must go to read and then use the correct cited reference. You
cannot just list Wikipedia.

Microbiology Lab Manual


All cited works should be listed here and formatted following the APA (American Psychological
Association) You can view these guidelines and sample references here:
http://owl.english.purdue.edu/owl/resource/560/1/. In the lab report, you will have used information from
other sources, and it is essential to use in text citations to accredit other researchers. Most of the introduction,
and much of the discussion, involve building upon the research of others. It is expected to quote the work of
others and, in fact, it is essential that you do so. Occasionally, you will use direct quotes from another source,
but most of the time you will be paraphrasing the work. You will need to create a bibliography or reference
list of all of the sources that you use, but you will also need to indicate within the text where your information
came from. Referencing is an essential part of writing any research paper. Common knowledge does not need
to be referenced.

Microbiology Lab Manual


Micro Lab Report Rubric
Criteria Does not meet Substantially meets Fully meets
expectations expectations expectations
1 2 3
Introduction Two or more components Includes all Correctly
Includes all of the following: of the introduction are components of the identifies Genus
Genus & species missing and/or incorrect introduction however species and all
Habitat at least 1 component is components of
Growth Requirements incorrect or the introduction
Mode of transmission Includes missing are included
Diseases, health issues components of the and correct
associated with organism introduction
Environmental issues /
concerns if related
Material & Methods Missing one or more item Missing one Lists all
Materials from the materials list component from the materials used
List of all materials used AND two or more of the materials list and/or in a table
Methods: tests performed are one of the tests format AND
Tests performed in order of incorrect and/or rationale performed or the lists all the tests
performance and purposes of for the selected tests is rationale given for the performed
tests explained inaccurate selected test in along with the
inaccurate purpose of each
test
Results Two or more of the Any one aspect of the Table includes
Results in table format to results table is incorrect results table is all tests
include for the identified incorrect OR performed and
Test performed, observations organisms OR Dichotomous key is the observations
and interpretation of results Dichotomous key is inaccurate and the correct
(+ / -) missing. interpretation of
Dichotomous key reflecting each test.
correct sequencing of tests to
reach your definitive
bacterial choice
Discussion / Conclusion Missing 1 or more from Missing 1 component Discussion and
Discussion includes: each of the discussion of the discussion points conclusion
problems &/or issues and conclusion points or or 1 component of the include all
encountered in performing not including any conclusion points points listed
the tests and how this could discussion or conclusion
be a problem in identification not included or does not
of an organism relate to the organism
Inclusion/exclusion rationale named in the introduction
for choices made
Conclusion includes:
Application of methodologies
and results to real life
situations
Conclusion follows the data

Microbiology Lab Manual


Format
Typed and printed (in color Hand written report. Materials list or results
All formatting
IF including color pictures table in paragraph is correct
and/or charts) Numerous spelling form. including
Correct spelling, grammar and/or grammar errors. materials list
and correct notation of Genus A few spelling and and any tables
species Single letter grammar mistakes. and
Genus and species name is abbreviations used (i.e. S. dichotomous
spelled out in the introduction epi vs. Staph. epi) Headings present but in key.
(for example, incorrect order.
Staphylococcus aureus) Results, discussion or All headings
No single letter conclusion refers to are present.
abbreviations of Genus pictures of slides to see
names can be used. the color yet report Genus species
Charts / tables are fully printed in black and names match
legible white. throughout
All headings present report.
Follows formatting
Correct spelling
instructions
and grammar.
References No references cited other Only 1 additional Cited references
References correctly than textbook and/or lab reference in addition to are formatted
annotated and formatted manual or only the textbook and/or lab correctly
using the APA (American Wikipedia referenced in manual.
Psychological Association) addition to the textbook Contains at
Must contain at least 2 and/or lab manual. 2 or more references least 2
references in addition to the not cited in APA references in
textbook and/or lab manual. Citations missing from format. addition to the
Wikipedia is NOT a the report. textbook and/or
reference, you must go to the lab manual.
source article.
All references
are cited in the
report.

Microbiology Lab Manual


WORKSHEET
Please attach the worksheet to the back of your report when you submit it.

Names ____________ _______________________

Unknown Number

Streak Plate: Growth in 4 quadrants: (2 points)

Isolated colonies in fourth quadrant: (2 points)

Gram reaction: (1 point)

Morphology and arrangement on Gram stain: (1 point)


Biochemical tests
Correct Selection and Interpretation of Biochemical tests: (4 points)

Biochemical test

Test Purpose Observation Result

Unknown Genus and species, based on biochemical tests: _____________ (2 points)

Total unknown points _________________________________ (Out of 12)

Total unknown report points ____________ (out of 30)

Microbiology Lab Manual


PREPARATION OF YOGURT

Food Microbiology

OBJECTIVE:

1. To make yogurt.
2. To understand the effects of fermentation on food.

Microbes are important in food production including foods such as beer, wine, cheese, yogurt,
sour cream, buttermilk. Yogurt is produced by the fermentation of warm milk by Lactobacillus
bulgaricus or Lactobacillus acidophilus and Streptococcus thermophilus. These bacteria grow
at 40-45oC. The milk is modified as a result of anaerobic fermentation which produces a
mixture of organic wastes including lactic acid and various other byproducts. These curdle the
milk and solidify the proteins as well as giving yogurt its unique flavor. Food fermentation
involves the use of starter cultures that contain known organisms to carry out specific and
reproducible fermentation reactions.

Chemical Reaction in yogurt preparation

Specific starter bacteria are added to the milk. Bacteria ferment milk sugar lactose into lactic
acid. The presence of the acid lowers the pH of the milk, making it acidic. As the pH is lowered,
the proteins casein in the milk begins to coagulate, giving firm texture to yogurt.

Yogurt is the best known food to contain healthy bacteria. Commercial yogurts are now being
enhanced with probiotics specifically to aid intestinal function to produce healthy digestion.
Yogurt is a concentrated protein product that has been enriched by additional enzymes and
vitamins produced by the microbes.

MATERIALS:
1. 1% milk 100 ml
2. One teaspoon of yogurt to use as starter culture
3. Steaming apparatus (Bunsen burner, ring stand, beaker and wire gauze)
4. Plastic container with lid
5. Spoon
6. Thermometer
PROCEDURE: Work in group
1. Label plastic container with your station number.
2. Put milk in the beaker and heat it up to 700 C to kill any bacteria present.
3. Add a teaspoon of starter culture in the plastic container.
4. Cool milk to 40- 500 C.
5. Add milk to plastic container and stir it.
6. Close plastic container with lid and incubate it at 400 C for four to six hours.

Microbiology Lab Manual


QUESTIONS:

1. Define probiotics

2. List the fermenting bacteria used to make yogurt.

3. Write the chemical reaction in yogurt preparation.

Microbiology Lab Manual


PHYSICAL CONTROL OF MICROORGANISMS
OBJECTIVES:

1. Name various types of physical methods used to control microbial growth.


2. Discuss the basic mechanisms involved in killing microorganisms using each method.
3. Discuss the mechanism of action of heat and ultra-violet light to kill microorganism.
4. Analyze the results of ultra-violet light experiment.

There are a variety of physical methods for control of microorganisms. Direct heat is used when
wire loops are flamed. Pressurized steam is used in autoclaves to sterilize a wide variety of
items, from microbiological media to linens. Ultra-violet light is used to sterilize air and
surfaces. Filters are used to sterilize liquids like antibiotics and vaccines which cannot be
heated. In this lab we will look at the effects of heat and ultra-violet light on different organisms.

HEAT:
Boiling water is effective in destroying organisms. This moist heat method of sterilization
causes conformational changes in proteins. Once enzymes or structural proteins change shape
the function is lost, therefore, the organisms die. Some bacteria are more sensitive to heat than
others. In this experiment organisms will be exposed to heat for different amounts of time. The
survivors will eventually grow in the broth. We will look for survivors by examining broth
cultures for turbidity.

ULTRA-VIOLET LIGHT:

Ultra-violet light at 265 nanometers causes the production of thymine dimers in the DNA of the
microorganism. These dimers prevent the transcription of the DNA into a viable mRNA.
Therefore protein synthesis is also interrupted which leads to lethal mutation. Without protein
synthesis organisms die. Sometimes the mutation is not lethal in which bacteria replicate but
only one characteristic of the organism is altered. We will examine the effect of U.V. light on
Serratia marcescens.

ATCCTTAGTTACG
TAGGAATCAATGC

UV

ATCCTTAGTTACG
TAGGAATCAATGC

Damage to DNA by UV light exposure

Microbiology Lab Manual


MATERIALS:
1. ultra-violet lamp
2. nutrient agar plates (TSA) - 4 plates for each organism
3. sterile swabs
4. cultures of Serratia marcescens.

PROCEDURE: Work in groups of 2

PROCEDURE: Work in groups of 2


1. Label a set of plates as 10 sec., 20 sec., 30 sec. without lid and one plate 30 sec.
lid on along with organism name, and your station number.
2. Make a lawn of bacteria on each plate with cotton swab.
3. Take the lids off of all the plates before irradiating except for the plate labeled as
30 sec. lid on.
4. Irradiate the plates for the appropriate time. (The lamp should be 6 inches from
the plates).
5. After irradiation put the lids back on all plates.
6. Place the plates in the petri plate wreck.
7. Incubation at 37 degrees Celsius for 48 hours.
8. Examine the plates for numbers and types of colonies.

Questions
1. Name different physical method used to control growth of microorganisms.

2. How does heat kills microorganisms?

3. What is the mode of action of Ultra-violet light?

4. What is the difference between lethal and non-lethal mutation?

Microbiology Lab Manual


CHEMICAL CONTROL OF BACTERIA

OBJECTIVES:

1. Define antiseptic and disinfectant.


2. Discuss the purpose of Antibiotic sensitivity test.
3. Perform the Standardized Disk Susceptibility Test against a Gram positive organism and
a Gram negative organism.
4. Interpret the test
5. Define zone of inhibition.
6. Discuss why this test is a quantitative and not a qualitative test

Many chemical agents inhibit bacteria. Antiseptics are used on living tissue to kill
microorganisms. Disinfectants are used on inanimate objects to remove and inactivate
organisms. A wide variety of commercial products that will inhibit microorganisms are
available.

ANTIBIOTICS
Antibiotics are natural substances produced by one microorganism (primarily molds) that inhibit
another organism or group of organisms (primarily bacteria). Sulfonamides were first used in
1935 followed by penicillin in 1940. Since then many useful antibiotics have been discovered,
such as chloramphenicol, tetracycline, and streptomycin.
The effectiveness of antibiotics is determined by the Standardized Disk Susceptibility Test, also
known as the Kirby-Bauer Method of Antibiotic Sensitivity Testing. A special medium called
Mueller-Hinton agar is used for antibiotic sensitivity testing because it gives reproducible
results and does not inhibit Sulfonamides. The agar is inoculated with a lawn of bacteria. Paper
discs containing known amounts of antibiotics are placed on the plates. After incubation, the
plates are observed for zones of inhibition of growth of the organism. The size of the zone of
inhibition is measured and compared to a table of known sizes of zones. The sensitivity of the
bacterium to a particular antibiotic can be determined. This method is quantitative due to the
fact that a standardized medium is used and the paper discs contain a known concentration of
antibiotic.

Mueller Hinton Agar

Microbiology Lab Manual


MATERIAL:

Antibiotics Sensitivity Testing


1. Mueller-Hinton agar plates (one per person)
2. selected antibiotic discs
3. sterile cotton swabs
4. forceps
5. cultures of E. coli and Staph. epidermidis

PROCEDURES: Work in pairs.

Antibiotics Sensitivity Testing


1. Label Mueller- Hinton agar plate with the name of the organism and your station
number.
2. Make a lawn of bacteria on each plate with cotton swab.
3. Dispense antibiotic discs on your plate with an antibiotic disc dispenser.
4. Invert the plates and incubate them at 37 degrees Celsius for 48 hours.
5. Measure the zone of inhibition for each antibiotic on each organism.
6. Determine if the organism is resistant, intermediately susceptible, or susceptible
to the antibiotic by comparing the zone of inhibition in mm to the standardized
chart of Zones of Inhibition.

Microbiology Lab Manual


Kirby-Bauer Test Results
Zone Size Interpretive Chart For The Kirby-Bauer Test

Diameter of Zone of Inhibition


Antibiotic Disc Code Resistant Intermediate Sensitive
(mm or less) (mm) (mm or more)
Ampicillin
Enterobacteriaceae 13 1416 17
Staphylococci 28 29
Haemophilus spp. 18 1921 22
S. pneumoniae AM10g
Streptococcus other than S. 18 1925 26
pneumoniae
Enterococcus spp. 16 17
Cephalothin
Enterobacteriacae and CF30 g 14 15-17 18
staphylococci
Erythromycin
Staphylococcus spp. & 13 14-22 23
enterococci E-15 g
S. pneumoniae & other 15 16-20 21
Streptococci
Gentamycin
Testing enterococci GM-120 g 6 7-9 10
Enterobacteriacae GM-10 12 13-14 15
Trimethoprim/ 1.25 g
Sulfamethoxazole 23.75 g
SXT
Enterobacteriacae, 10 11-15 16
Staphylococci
Tetracycline
Enterobacteriacae, 14 15-18 19
Staphylococci Te-30
S. pneumoniae & other 18 19-22 23
Streptococci

Microbiology Lab Manual


Questions
1. Difference between antiseptics and disinfectants.

2. What is a zone of inhibition?

3. How is zone of inhibition measured in Kirby-Bauer Method of Antibiotic Sensitivity test?

4. Kirby-Bauer Method of Antibiotic Sensitivity is quantitative or qualitative test?

Microbiology Lab Manual


FUNGI
OBJECTIVES
1. Define fungi.
2. Discuss general characteristics of fungi.
3. Classify fungi based upon morphology and based upon sexual and asexual spores.
4. Identify sexual and asexual spores under the microscope.
5. Name the human diseases caused by fungi.

I. GENERAL

A. Fungi are chemoheterotrophs that require organic compounds for energy and carbon. Their
cell wall is made of a polysaccharide chitin. They are absorptive heterotrophs that they secret
exoenzymes into the environment, and then absorb the digested nutrients.
Majority are saprophytes that decompose dead or decaying organic matter. Some are
parasites of humans, animals, and plants.

B. Includes Molds ( filamentous), Yeasts ( unicellular) and dimorphic (both mold and yeast)

Division into these categories is based on overall appearance and how they grow.

C. Nutritional Adaptations

These characteristics allow fungi to grow on unlikely substrates such as painted walls
and shoe leather.

1. Fungi usually grow better in an acidic pH (5.0) with high sugar concentrations.

2. Most Molds are aerobic, so they grow on surfaces rather than through a substrate. Yeasts
are facultative anaerobes.

3. Most fungi are more resistant to osmotic pressures than bacteria are; most fungi are
therefore able to grow in high sugar or salt concentrations.

4. Fungi are capable of growing on substances with very low moisture content, generally
too low to support the growth of bacteria.

5. Fungi require somewhat less nitrogen for growth than bacteria.

6. Fungi are capable of using complex carbohydrates, such as wood, that most bacteria
cannot metabolize.

D. Fungi are found in almost any climate but prefer warm, moist environments.

Microbiology Lab Manual


II. CHARACTERISTICS OF FUNGI

Yeast: (Candida albicans and budding yeast)

1. They are non-filamentous unicellular fungi that are typically spherical or oval.

2. They are widely distributed.

3. Reproduce by Budding parent cell forms a protuberance (bud) that elongates, the
parent cells nucleus divides and one nucleus migrates into the bud. Cell wall material
laid down between bud and parent cell, bud breaks off.

Molds (multicellular)-

1. The body (thallus) consists of long filaments of cells joined together called
hyphae (hypha).

2. Hyphae grow by elongating tips. Hyphae grow, intertwine and form a mass called
mycelium.

a. Vegetative mycelium attached to surface of substrate, concerned with


obtaining nutrients.

b. Aerial mycelium projects upwards away from surface (fuzzy part),


reproductive portion.

3. Fungi reproduce both sexually and asexually.

Dimorphic Fungi

1. Some fungi (most notably the pathogenic species) exhibit dimorphism.

2. Dimorphic fungi have both Mold and Yeast life cycle stages.

3. Dimorphism is frequently temperature dependent.

a. Grow mold like with vegetative and aerial mycelia at 25 degrees C.

b. Grow yeast like at 37 degrees C.

Microbiology Lab Manual


III. Medium for Growth of Fungi

A. Sabourauds Dextrose Agar

1. favors fungal growth over bacteria


2. low pH (5.6) and high sugar concentration (inhibits most bacteria)

IV. Reproductive Structures


Fungal life cycles are usually complex, involving both sexual and asexual forms of
reproduction. Spores are formed from the aerial mycelium and can be asexual or sexual.

A. Zygomycota conjugation fungi

1. Can reproduce either sexually or asexually.


2. Genus Rhizopus
3. Asexual reproduction by sporangiospores.

These are asexual spores formed within a sac (sporangium) at the end of aerial
hyphae called sporangiophores. A sporangium can contain hundreds of
sporangiospores.

4. Sexual reproductive spores called zygospores.

Microbiology Lab Manual


When the nuclei of two cells that are morphologically similar to each other fuse and
form a zygote, the only diploid cell in the fungal life cycle; undergoes meiosis and
returns to haploid state. The zygospore begins to form when 2 hyphae of 2 similar
fungi fuse. The cells involved in the fusion have their genetic material combined to
form a diploid zygote that is inside the zygospore. The zygote undergoes meiosis to
return to the haploid state.

B. Ascomycota Sac Fungi


Ascospores are produced in a sac like structure called an ascus. There are usually
eight (8) ascospores in an ascus.

B. Basidiomycota Club fungi- Common name is derived from the shape of the
basidium that bears the sexual basidiospores; includes mushrooms.

Microbiology Lab Manual


Sexual spore formed externally on a base pedestal called a basidium. Usually
have four basidiospores per basidium.

D. Deuteromycota Fungi Imperfecti

1. They are imperfect because they have not yet been shown to produce sexual spores.
(Asexual reproduction only)

Spores formed at the end of hyphae are called conidia.

2. Genus Aspergillus and Penicillium reproduce via conidiospores. A conidiospore is


a unicellular or multicellular spore that is not enclosed in a sac.

Conidiospores are produced in a chain at the end of a conidiophore.

Microbiology Lab Manual


Major groups of Representative Genus Sexual Spore Asexual Spore
Fungi
Zygomycota Rhizopus Zygospore Sporangium
Bread molds sporangiospore

Ascomycota Schizosaccharomyces Ascospore Conidium


Sac Fungi conidiospore
Basidiomycota Coprinus Basidiospore Conidia
Club Fungi conidiospore
Deuteromycota Candida (yeast) None Conidia
Imperfect Fungi Aspergillus (mold) Conidiospore bud
No sexual stage Penicillium

Fungal diseases
Name Mode of transmission Disease
Blastomyces dermatitidis Inhalation of dust carrying Cutaneous blastomycosis; Pulmonary
fungal spores blastomycosis
Trichophyton spp. spores shed in the Dermatophytoses
Microsporum spp., environment by infected Tinea pedis "athletes foot"
Epidermophyton individuals Tinea cruris " jock- itch"
Tinea unguium
Tinea capitis
Tinea barbae
Malassezia furfur Normal microbiota on skin Tinea versicolor (Pityriasis versicolor);
dandruff; seborrheic dermatitis
Sporothrix schenckii Thorn pricks, wood splinters Sporotrichosis "Rose-Gardners disease"
Aspergillus spp. contamination from spores Aspergillosis
Carcinogenic mycotoxin causes
hepatocellular carcinoma
Cryptococcus neoformans Inhalation of spores from Cryptococcal meningitis in AIDS patients.
contaminated pigeon or
chicken droppings
Coccidioides immitis Inhalation of spores Coccidioidomycosis (Valley fever)
Histoplasma capsulatum Inhalation of spores near Histoplasmosis (Spelunker's disease)
bird droppings
Pneumocystis jirovecii Inhalation of spores Pneumocystis pneumonia in AIDS patients.
(carinii)
Candida albicans opportunistic infection, oral thrush, vaginal thrush, Candidal
sexually transmitted onychomycosis, Candidal paronychia, Diaper
candidiasis, Congenital cutaneous
candidiasis, Perianal candidiasis,
Piedra "Trichosporosis" Fungal growth on hair

Microbiology Lab Manual


Questions
1. Describe general characteristics of fungi.

2. What is the role of fungi in ecosystem?

3. Describe morphological classification of fungi.

4. Difference between Vegetative and Aerial mycelium.

5. Name the agar on which fungi can grow.

6. Name the sexual and asexual spores of.


Sexual Asexual
a. Zygomycota
b. Ascomycota
c. Basidiomycota
d. Deuteromycota

6. List three genera which cause dermatophytoses.

Microbiology Lab Manual