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:

  • Aeromonassalmonicida

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:

  • Aeromonassalmonicida
  • 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
  • Aeromonassalmonicida

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.
  1. Incubate at 15°C for 7 days.
  2. 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

  • Aeromonassalmonicida– 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

  • Polymyxin B
  • Oxytetracycline
  • Erythromycin
  • Neomycin Sulfate

Procedure

  1. Work with a partner.
  2. Label the TSA plate Aeromonassalmonicida 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 Aeromonassalmonicida and completely coat the TSA plate (covering the entire surface area) labeled Aeromonassalmonicida.
  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

CytochromeOxidase 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.
  4. Disinfect a slide with methanol and wipe dry. Use the same slide for both Plate A and Plate B.
  5. Place one drop of 3% KOH solution on slide.
  6. Use a toothpick to remove a portion of a single colony from Plate A agar plate.
  7. Swirl bacteria in drop of KOH (up to 60 seconds). Record results.
  8. Perform steps b through d for Plate B.
  9. Perform cytochromeoxidase test.
  10. Disinfect a slide with methanol and wipe dry. Use the same slide for both Fish A and Fish B.
  11. Place one drop of cytochromeoxidase solution on slide. (May place slide on white paper to make results easier to interpret.)
  12. Use a toothpick to remove a portion of a single colony from Fish A agar plate.
  13. Swirl bacteria in drop of cytochromeoxidase solution (up to 15 seconds). Record results.
  14. Perform steps b through d for Fish B.
  15. Perform motility test for Plate A and Plate B. Record results.
  16. Using the flow chart, record your presumptive identification of the bacterial species.