Facilitator Guide for Basic Culture Media and Isolation

Techniques Laboratory Exercises

Introduction

The Basic Microbiology Curriculum: Basic Culture Media and Isolation Techniques Course is a
blended learning activity that includes both eLearning and hands-on laboratory exercises.
Both components of the course are equally important in providing knowledge and actual
laboratory experience to the participant. This facilitator guide is meant to serve as a manual
for the supervisor/mentor that will be overseeing the completion of the laboratory exercises
after the eLearning activity has been completed. The manual contains instructions for the
overall laboratory exercise components, objectives, laboratory setup, a supply list, laboratory
exercises, instructions and answer key as well as job aids.

The goal of these exercises is to allow the participant to use the information and procedures
learned during the eLearning portion of the course and apply them using hands-on laboratory
exercises. Please note: These laboratory exercises may be edited according to your
laboratory’s standard operating procedures or guidelines, if necessary. The job aids and
laboratory exercises were created with the forethought that laboratory procedures may vary
from laboratory to laboratory and therefore, may need to be edited according to the
procedures or protocols followed within that laboratory.

The participant of the course is strongly recommended to complete the laboratory exercises
to transfer the didactic content of the course to experiential knowledge gained through hands-
on laboratory exercises with the equipment from their laboratory. The supervisor/mentor
should work with the participant to develop these laboratory skills as well as confirm that
these exercises have been completed. The number and types of exercises completed will be
at the discretion of the supervisor/mentor based on procedures followed within their
laboratory. After the laboratory exercises are completed and discussed with the
supervisor/mentor, the supervisor/mentor should then follow-up the exercises with instruction
related to your laboratory’s specific procedures or guidelines.

Laboratory Training Branch

Facilitator Guide for Basic Culture Media and Isolation
Techniques Laboratory Exercises, Page 2

Laboratory Exercise Objectives

After completing the laboratory exercises, the participant will be able to:
• Explain the different types and forms of culture media.
• Correlate culture media with specimen type.
• Describe the critical steps in the inoculation of media to obtain isolated colonies.
• Identify commonly encountered problems with culture media and isolation techniques.

Initial Planning for the Laboratory Exercises

1. Communicate with the participant and schedule days/times to complete the laboratory
exercises.
2. Collect the supplies and culture media necessary to complete the exercises (see supply
and culture media list).

Day(s) of Scheduled Laboratory Exercises

3. Set up supplies for the exercises.
4. Remind the participant about the use of proper PPE and laboratory equipment according
to your laboratory’s procedures and safety manual.
5. Pull culture media out of the refrigerator (if necessary) to come to room temperature.
6. Participant should have a copy of the laboratory exercises and job aids as a printout from
the eLearning course.
7. Have participant complete each exercise with your approval. Please feel free to instruct
participant as they work or after the exercise is completed. Exercises may be completed
all at once or as time permits.
8. Relay to the participant any information that is needed to comply with your laboratory’s
standard operating procedures (SOPs) or safety procedures.

Laboratory Training Branch

Facilitator Guide for Basic Culture Media and Isolation
Techniques Laboratory Exercises, page 3

Supply List
1. Personal protective equipment (PPE) and laboratory equipment
2. Loops (sterile plastic or metal)
3. Incinerators or Bunsen burner (if using metal loops)
4. Broth culture containing isolated colonies
5. Labelling pen
6. Biohazard waste container: for personal protective equipment
7. Sharps container: For loops
8. Incubator

Culture Media List

9. Broth culture containing microorganisms to be plated (any positive culture will work)
10. Culture media: BAP, CHOC, MAC, C-CNA/PEA, CAMPY, CHROMagar, HE/XLD, SMAC,
AnaBAP
11. Previously inoculated media that had problems (to be used for demonstration and problem
solving)

Laboratory Training Branch

Facilitator Guide for Basic Culture Media and Isolation
Techniques Laboratory Exercises, page 4

Laboratory Exercise I

After completing this laboratory exercise, the participant will be able to:
• Explain the different types and forms of culture media.
• Correlate culture media with specimen type.

Exercise 1
Choose culture media appropriate for the specimen source and label the media.
• Give the participant a broth culture containing microorganisms of your choice. Provide
information to the participant on where the specimen was isolated from (such as a wound,
respiratory or stool). Ask the participant to plate the media based on the specimen source
of the culture. Ask he/she to label their plates with name, source, and date.

Notes
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
________________________________

______________________
Mentor/Supervisor /Date

Laboratory Training Branch

Facilitator Guide for Basic Culture Media and Isolation
Techniques Laboratory Exercises, page 5

Laboratory Exercise II

After completing this laboratory exercise, the participant will be able to:
• Describe the critical steps in the inoculation of media to obtain isolated colonies.

Exercise 1
Perform quadrant streaking to obtain isolated colonies using the quadrant streaking procedure
on the job aid or your laboratory standard operating procedure.
• With the broth culture given to the participant in exercise one, and the labeled plates, have
the participant perform quadrant streaking on each type of media.
• The participant should be able to follow the Quadrant Streaking job aid (See Appendix) to
perform isolate streaking.
• After streaking the culture plates, have the participant invert the plates and incubate at the
required temperature for 18-24 hours.

Notes
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
________________________________

______________________
Mentor/Supervisor /Date

Laboratory Training Branch

Facilitator Guide for Basic Culture Media and Isolation
Techniques Laboratory Exercises, page 6

Laboratory Exercise III

After completing this laboratory exercise, the participant will be able to:
• Identify commonly encountered problems with culture media and isolation techniques.

Exercise
Observe culture media plates for commonly encountered problems that may arise.
• The participant should examine culture media plates from Laboratory exercise II that lacked
isolated colonies after streaking in the four quadrants. The observations seen and the
possible solutions should be discussed with the supervisor/mentor.
• Provide the participants with at least one culture media plate that had water on the media
when it was streaked.
• Provide an example of growth of a microorganism not on the line of streaking on the culture
plate.
• Provide the participants with at least one culture plate that has dried up.

Notes
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
________________________________

______________________
Mentor/Supervisor /Date

Laboratory Training Branch

Culture Media Table

Introduction
Culture media is useful to isolate and identify microorganisms from various sites in the human
body. This culture media table will help you learn about the various general media used in a
public health or clinical microbiology laboratory. The table has basic culture media you will use
as you process a specimen in the laboratory or work with an isolate sent to you from a clinical
or reference laboratory.
Media Enriched Selective Differential
Anaerobe Blood Agar (AnaBAP) X

Blood Agar (BAP) also called Sheep Blood Agar (SBA) X X

Campylobacter Blood Agar (CAMPY) X X

Chocolate Agar (CHOC) X

CHROMagar or Chromogenic Agar X X X

Columbia Colistin Nalidixic Acid Agar (C-CNA or CNA) X X X

Gram Negative (GN) Broth/Selenite F (SF) Broth X X

Hektoen Enteric Agar (HE) X X

MacConkey Agar (MAC) X X

Mac-Sorbitol (Sorbitol-MAC) for E. coli 0157 Agar

X X
(SMAC)

Phenylethyl Alcohol Agar (PEA) X X X

Salmonella-Shigella Agar (SS) X X

Thayer-Martin/Martin Lewis Agar (TM/ML) X X

Thioglycollate Broth (THIO) X

Thiosulfate Citrate Bile Salts-Sucrose Agar (TCBS) X X

Xylose Lysine Deoxycholate Agar (XLD) X X

Laboratory Training Branch

Specimen Type and Culture Media Table

Introduction
Specimens received in the laboratory must be plated on appropriate culture media to isolate
the microorganisms that are suspected of causing infection. The selection of culture media to
plate is based on the site of the infection and the suspected microorganism causing the
infection. The table below provides examples of recommended media that can be plated to
recover microorganisms from a particular body site. Selection of types and forms can vary
depending on the laboratory protocol and availability.

Specimen Type Culture Media Used

Cerebral Spinal Fluid (CSF) BAP, CHOC
BAP, MAC, HE or XLD*, CAMPY, GN Broth or SF
Gastrointestinal Tract Broth*, SMAC, CHROMagar**, TCBS (Vibrio spp.),
SS*
Genital BAP, CHOC, TM/ML*
Respiratory Tract BAP, CHOC, MAC, CHROMagar** (MRSA)
Tissue BAP, CHOC, MAC, C-CNA or PEA*, THIO, AnaBAP***
Urine
*Some of these media perform the same BAP, MAC , and
function CHROMagar**
one or more may be used depending
on a laboratory’s
Wound or Abscessprotocols. BAP, CHOC, MAC, C- CNA or PEA*, AnaBAP***
** CHROMagar formulations vary according to their use. CHROMagar for stools differs from
that used to detect MRSA or urinary tract pathogens.
*** AnaBAP may have different formulations that are used based on a laboratory’s protocols.
Legend
• AnaBAP = Anaerobic Blood Agar
• BAP = Blood Agar
• CHOC = Chocolate Blood Agar
• CAMPY = Campylobacter Agar
• C-CNA (CNA) = Columbia Colistin Nalidixic Acid Agar
• CHROMagar = Chromogenic Agar
• GN Broth or SF Broth = Gram Negative Broth/Selenite F Broth
• HE = Hektoen Enteric Agar
• MAC = MacConkey Agar
• PEA = Phenylethyl Alcohol Blood Agar
• SMAC = MacConkey-Sorbitol (Sorbitol-MacConkey) Agar
• SS = Salmonella Shigella Agar
• TCBS = Thiosulfate Citrate Bile Salts-Sucrose Agar
• THIO = Thioglycollate Broth
• TM/ML = Thayer-Martin Agar or Martin Lewis Agar
• XLD = Xylose Lysine Deoxycholate Agar

Laboratory Training Branch

Quadrant Streaking for Isolation

Introduction
Quadrant streaking an agar plate is performed to obtain isolated colonies from a specimen.
Isolated colonies are achieved by performing a four quadrant streak which results in a dilution
of the inoculum from one quadrant to the next. Typically, by the third or fourth quadrant
isolated colonies are present and are used for further testing.

Supplies
1. Personal protective equipment
2. Specimen to be streaked
3. 1.0 – 10ul metal or sterile plastic loops
4. Incinerator (if using metal loops)
5. Sharps biohazard waste container
6. Labeling pen
7. Sterile agar plate

Instructions
1. Label the bottom of the agar plate 1
2. Use a sterile loop to remove a small amount of
bacterial growth (from broth culture or a colony
2
from solid media)
Note: If you are using a metal loop, sterilize it in 4
the incinerator for 5 to 10 seconds before
streaking each quadrant. Allow the loop to cool
before streaking. You can make sure the loop is
cool by touching an uninoculated area of the 3
agar.
3. Inoculate the first quadrant by making an one
inch streak down, then streaking back and forth 6. Turn the plate another quarter turn
across this inoculum 7. Use a sterile loop and repeat the process
4. Turn the plate a quarter turn above but only go into the second
quadrant two or three times and
5. Use a sterile loop to streak the second quadrant
complete the third quadrant streak
by going through the edge of the first quadrant
approximately four times and then continue 8. Turn the plate another quarter turn
streaking the second quadrant without going 9. Use a sterile loop and streak the fourth
back into the first quadrant again quadrant the same as the third but let
streaks get smaller and trail off
10. Replace the lid on the agar plate and
incubate according to your laboratory’s
testing protocol

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Laboratory Training Branch