Fish 424
Laboratory Exercises (Lab 7& 8)
Bacteriology
Introduction
Exercise #1: Bacterial Streak for Isolation (Lab 7 & 8)
Exercise #2: Bacterial Motility (Lab 7)
Exercise #3: Colony and Cell Morphology (Lab 7)
Exercise #4: Necropsy (Lab 7 & 8)
Exercise #5: Antibiotic Sensitivity (Lab 7 & 8)
Exercise #6: Unknown diagnostics (Lab 8)
EXERCISE #1: Streak for Isolation
Introduction
One of the first steps in identifying a possible finfish disease agent and
probably the most important technique is obtaining a PURE CULTURE. This is
commonly performed by the preparation of a streak plate. This will allow you to
isolate individual bacterial colonies. To properly streak a plate for isolation
means spreading out the organism(s) by means of the inoculating loop until
single colonies result. Each colony consists of numerous cells that originate by
cell division from one bacterial cell. In other words, each colony represents a
pure culture of bacteria.
Materials
Inoculating loop
TSA media plates
Broth culture of:
 Aeromonas salmonicida
Procedure
1. After observing the correct the procedure for streaking a plate from the
instructor and referring to the diagram, prepare a streak plate of the
bacterial cultures from the broth cultures.
2. Label the plate with your name and the name of the bacteria.
3. Seal with parafilm, invert, and place plates in the incubation box
4. Incubate at 15°C for 7 days.
5. After 7 days, record your observations. Were you able to get isolated
colonies? Were there any other types of bacterial contamination on your
plate?
EXERCISE #2: Bacterial Motility
Introduction
This procedure is routinely used for the detection and identification of
certain finfish pathogens and can be found in the flow chart for presumptive
identification of bacteria (handout). Some bacteria possess one or more very
fine threadlike appendages called flagella which aid in locomotion. Observing a
bacterial sample using the wet mount procedure will identify whether the
organism is motile or non-motile. Motile bacteria appear to be freely moving
around when viewed under the microscope whereas non-motile bacteria remain
motionless. It is important to note that Brownian motion occurs under most wet
mount procedures and should not be misinterpreted as positive motility.
Brownian motion is a peculiar dancing motion exhibited by finely divided particles
and bacteria in suspension brought on by the excitation of heat from the light
source of the microscope.
Materials
PBS
Glass slide
Immersion oil
Cover slips
Agar plate cultures of:
 Aeromonas salmonicida
 Flavobacterium psychrophilum
Procedure
1. Place a drop of 0.85% saline onto a glass slide.
2. Using an inoculating loop remove a portion of a colony from a media plate
and resuspend bacteria in the saline drop.
3. Cover with sample with a cover slip.
4. View under oil immersion (100X).
5. Record the motility of each specimen.
EXERCISE #3: Colony and Cell morphology
Objectives
 Describe colony morphology, bacterial shapes, and cell arrangement
 Prepare bacterial smears
 Perform a Gram stain of bacterial smears
Materials
Agar plate cultures of:
 Flavobacterium psychrophilum
 Aeromonas salmonicida
Gram stain reagents
Inoculating loop
Bunsen burner
Glass slides
Microscope
Immersion oil
Procedure
1. Using the colony morphology handout describe the colonies on the agar
plates and record in your notebook. Do this for each sample.
2. After observing the correct procedure from the instructor, perform a
bacterial smear onto glass slides of each bacterial species from the
assigned plates.
3. In preparation of smears, a small amount of bacteria from a colony is
spread on the surface of a clean slide. The bacterial smear is first allowed
to air dry and is then passed through a flame. This procedure has two
functions: 1) it kills the bacteria and 2) it denatures the proteins in the cells
thereby fastening or FIXING the bacteria to the slide. IMPORTANT: do
not overheat the smear while running slide over flame. You should be
able to hold the glass slide without burning your fingers. Overheating your
sample can destroy the bacteria and it will appear shapeless or enlarged.
4. Perform a Gram stain of bacterial smears. Blot dry with a paper towel.
5. Examine the slides under the oil immersion lens (100X) of the microscope
& record your observations.
EXERCISE #4: Necropsy
Introduction
The idea behind this exercise is to gain experience isolating bacteria from the
kidney and spleen of fish. Typically, a fish health diagnostic technician will test
the kidney, spleen, and any other organ that has visible lesions.
Materials
1. Necropsy kit
2. TYES plate
3. Fish
4. Beaker of 95% ETOH
Procedure
1. Work with a partner (1 fish per group).
2. Obtain a TYES plate and divide it into three parts. Label the plate with
your name and date and each of 3 triangles kidney, liver or spleen.
3. Disinfect your clean necropsy tools in ETOH.
4. Obtain a fish and aseptically remove a portion of the kidney.
5. Smear the kidney on the portion of the plate labeled “kidney”.
6. Using an aseptic loop, streak the tissue for isolation.
7. Perform the same procedure with the spleen and liver.
8. Seal plate with parafilm, invert, and place in the incubation box.
5. Incubate at 15°C for 7 days.
6. After 7 days, record your observations. Did you observe more than one
colony type in the kidney or spleen of either fish? Describe the colonies
that you observed (use terms from Exercise 3).
EXERCISE #5: Antibiotic sensitivity
Introduction
Antibiotic sensitivity screening of isolated bacterial pathogens from
infected fish can be a very valuable tool for determining possible treatment
strategies. Antibiotics can be delivered to fish by mixing into feed or by
immersing the fish in a static bath containing the drug. But knowing which
antibiotic is the most successful candidate for treatment needs to be performed
first. The disc-plate technique can be used to determine the susceptibility of the
microorganism to various antibiotics.
Objective
Gain experience in performing the disc-plate technique and interpret
susceptibility by measuring the zone of inhibition.
Materials
 Aeromonas salmonicida – saline broth tube
 Flavobacterium psychrophilum – saline broth tube
 2 sterile cotton swabs
 1 TSA plate
 1 TYES plate
 Beaker of 95% ETOH
 Necropsy kit
 Antibiotic discs
o Polymyxin B
o Erythromycin
o Oxytetracycline
o Neomycin Sulfate
Procedure
1. Work with a partner.
2. Label the TSA plate Aeromonas salmonicida and label the TYES plate
Flavobacterium psychrophilum. Also include your name, the date, and
media type (TSA or TYES).
3. Aseptically dip the sterile cotton swab in the broth tube containing
Aeromonas salmonicida and completely coat the TSA plate (covering the
entire surface area) labeled Aeromonas salmonicida.
4. Discard the cotton swab in the bag provided. Please be careful not to
contaminate the counter tops with your sample.
5. Disinfect your forceps in ETOH, air dry, and place all four antibiotic discs
onto the surface of the agar. Be sure to place the discs equal distance
from each other.
6. Repeat this procedure with the TYES plate for Flavobacterium
Psychrophilum.
7. Seal with parafilm and place plates in the incubation box. Do not invert
agar plate.
8. Incubate at 15°C for 7 days and record your results. Which antibiotic
would be the best to be used against each of these bacterial pathogens?
Exercise #6: Unknown diagnostics
Materials
Culture plates from Plate A and Plate B
KOH test solution
Cytochrome Oxidase test solution
PBS
Toothpicks
Coverslips
Slides
Flow chart handout
Procedure
Read through the directions for each test before you begin.
1. Record presence/absence of pigment in media.
2. Record the color of the bacterial colony.
3. Perform KOH test.
a. Disinfect a slide with methanol and wipe dry. Use the same slide
for both Plate A and Plate B.
b. Place one drop of 3% KOH solution on slide.
c. Use a toothpick to remove a portion of a single colony from Plate A
agar plate.
d. Swirl bacteria in drop of KOH (up to 60 seconds). Record results.
e. Perform steps b through d for Plate B.
4. Perform cytochrome oxidase test.
a. Disinfect a slide with methanol and wipe dry. Use the same slide
for both Fish A and Fish B.
b. Place one drop of cytochrome oxidase solution on slide. (May
place slide on white paper to make results easier to interpret.)
c. Use a toothpick to remove a portion of a single colony from Fish A
agar plate.
d. Swirl bacteria in drop of cytochrome oxidase solution (up to 15
seconds). Record results.
e. Perform steps b through d for Fish B.
5. Perform motility test for Plate A and Plate B. Record results.
6. Using the flow chart, record your presumptive identification of the bacterial
species.
Plate A
Pigment in media
Colony color
KOH
Cytochrome oxidase
Motility
Identification
Plate B