HES 032- Microbiology and Parasitology

College of Nursing – RADL (AY 2021-2022)

Group Members:
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______________________________ ______________________________
______________________________ ______________________________

CULTURE MEDIA

I. OBJECTIVES: At the end of the laboratory period, the student shall be able to:
1. Identify and classify the different types of culture media.
2. Differentiate each according to its function or use, preparation, composition and
physical state.

II. RATIONALE
Microorganisms need nutrients and certain environmental conditions in order
to grow and reproduce. In the environment, these microorganisms have adapted to the
habitats most suitable to their needs. In the laboratory however, these requirements
must be met by a culture medium.
Culture media are substances used for the cultivation and preparations of
bacteria and related organisms. They are used for a variety of purposes in the laboratory.
Their performance must therefore be judged acceptable to ensure that they serve the
purpose for which they are prepared.
Culture media are used to propagate bacteria, fungi, viruses and in some cases,
animal parasites. Not only must a medium support the growth of an organism, but that
organism should exhibit typical colonial and microscopic morphology on the medium.
Variations in composition of the medium may alter these characteristics of organisms,
e.g., production of acid and gas in carbohydrate fermentation media or hemolysis of
erythrocytes in blood agar media.

The development of microorganisms upon the culture media is dependent upon

several important factors:
1. The proper food elements must be available.
2. Oxygen must be available as required.
3. A certain degree of moisture is necessary.
4. A medium must be of the proper pH reaction.
5. Proper temperature relations must prevail.
6. The medium must be sterile.
7. Contamination must be prevented.

Compiled by: CJBregente, RMT, MPH, MSc.

Southwestern University PHINMA, College of Medical Technology
 CLASSIFICATION OF CULTURE MEDIA
Culture media can be classified according to three primary levels:
1. Physical State 3. Functional Type
2. Chemical Composition

A. According to their Physical State

1. Solid Media – consist mainly of substances mixed in a solidified agar, gelatine or
albumin matrix. Agar is usually added in concentration of 2-3%. Such preparations
settle at about 38˚C. Gelatin is added in proportions of 1-1.5% and such media
solidify at temperature below 25˚C. Albumin unlike agar or gelatine coagulates with
heat, e.g., Loeffler’s coagulated serum medium and most of the solid media for the
cultivation of Mycobacterium tuberculosis.
2. Semi-solid Media – are especially used for keeping stocks of the bacteria for long
periods of time. Agar is usually added in amounts of 0.5-1.0%.
3. Liquid Media – are prepared without such substances as gelatine or agar.

B. According to their Chemical Composition

1. Synthetic Media – one in which the chemical composition of the ingredients is
known.
Examples: Ringer’s solution and Locke’s solution
2. Non-synthetic Media – one in which the precise composition of some or all the
nutritive elements used is not definitely known.
Example: Meat extract broth
3. Living tissue – one in which living tissue cells are present. These are especially used
for the cultivation of rickettsiae, Chlamydia and viruses.
Examples: HeLa cell lines, Primary Monkey Kidney cells and embryonated eggs

C. According to their Use/Functional Type:

Most media used in the clinical bacteriology laboratory belong to one of the following
types:

1. Simple Media – are ordinary media which are not enriched and in which non-
fastidious organisms are easily cultivated.
Examples: Nutrient broth and Nutrient agar

2. Specific Media – are special media which are used for the cultivation of a particular
organism.
Examples: Loeffler’s Coagulated Serum medium, Lowenstein-Jensen medium,
Saboraud’s Dextrose Agar and Fletcher’s medium

3. Enriched Media – include 5% sheep blood agar, enriched chocolate agar and
several infusion-based media, all of which may be further enriched by additives.
 HES 032- Microbiology and Parasitology
College of Nursing – RADL (AY 2021-2022)

These media must be carefully tested for sterility before they are used since even
fastidious organisms are likely to grow. Examples: Blood Agar, Chocolate Agar

4. Differential Media – include among others MacConkey’s and EMB agars, Triple
Sugar Iron and Mannitol Salt Agar, which contain dyes, sugars and indicators
designed to elicit a characteristic biochemical response (usually a color) and are
used to differentiate groups of organisms such as lactose fermenters and non-
lactose fermenters.

5. Selective Media – contain, in addition to components found in differential media,

agents designed to further inhibit most Enterobacteriaceae and to select from the
specimen those strains resistant to the higher concentrations of the selective
components. Some enteric media combine both selective and differential
characteristics. Hektoen enteric agar and xylose lysine desoxycholate agar may be
classed as selective because they are somewhat more inhibitory than MacConkey
selective media also are capable of detecting H2S production.
Examples: MacConkey Agar, Eosin Methylene Blue, Mannitol Salt Agar, Thayer
Martin Medium, Saboraud Agar, Hektoen Agar

6. Transport Media – are devised for prolonging the survival of microorganisms when
a significant delay occurs between collection and culturing.
Examples: Cary Blair Transport Medium, Amies Transport Medium

D. According to Preparation
1. Plate Media (Weigh à Autoclave à Dispense) [Except: SSA, TCBS]
2. Tube Media (Weigh à Dispense à Autoclave)

III. MATERIALS:
Sterile Petri dishes Sterile test tubes or Culture tubes
Different culture medium:
Tryptic Soy Agar/ Blood agar base Thioglycollate broth
MacConkey agar Simmon Citrate agar (SCA)
Triple Sugar Iron agar Sulfide Indole Motility agar (SIM)
Mueller Hinton agar Nutrient broth
Nutrient agar
Salmonella-Shigella Agar
Distilled water
Autoclave
Erlenmeyer flask

Compiled by: CJBregente, RMT, MPH, MSc.

Southwestern University PHINMA, College of Medical Technology
IV. PROCEDURE:

1. The laboratory instructor will assign a certain culture medium to your group.
2. The instructor will provide the package inserts or manufacturer recommendation of
the assigned culture media, or you may refer to the label indicated in the culture
media bottle as well. It is very important to follow the manufacturer’s directions
regarding the ratio and proportion of the agar to that of the diluents.
3. Dispense the culture media properly to their recommended storage.

References:
• Basit S.A & Juayang A.C. (2016). Outcomes-based education manual in clinical bacteriology. (1st Ed). Malabon,
Philippines. APD Educational Publishing House
• Delost, MD. (2015). Introduction to diagnostic microbiology for laboratory sciences. (1st Ed). Jones & Barlett
Publishers: Burlington, MA
• Ho, H., Roldan – Gan, R., Verbo, V. (2005). Microbiology and parasitology: laboratory manual for the health sciences.
Philippines: C & E Publishing Inc.

Multimedia resources:
1. Media preparation (Plate Method): https://youtu.be/cneascR3OEc
2. Agar Slant preparation: https://youtu.be/84rwtFQfAms
3. Liquid medium preparation: https://youtu.be/DyIXxjNbQbU
4. Blood agar preparation: https://youtu.be/7gFaRtqUWIA
5. Culture media: https://microbiologyinfo.com/category/culture-media/
6. List of culture media used in microbiology with their uses: https://microbiologyinfo.com/list-
of-culture-media-used-in-microbiology-with-their-uses/
 HES 032- Microbiology and Parasitology
College of Nursing – RADL (AY 2021-2022)

V. OBSERVATIONS
Provide pictures of the different culture media. Write the function of each culture
medium below.

A. Plated culture media

Blood Agar MacConkey Agar Chocolate Agar

Mueller Hinton Infusion agar Nutrient agar SSA

Purpose: Blood agar: ___________________________________________________________

MacConkey agar: _______________________________________________________
CAP: _________________________________________________________________
MHIA: ________________________________________________________________
Nutrient agar: __________________________________________________________
SSA: _________________________________________________________________

Compiled by: CJBregente, RMT, MPH, MSc.

Southwestern University PHINMA, College of Medical Technology
B. Tubed culture media

Thioglycollate broth Nutrient broth Triple Sugar Iron agar SIM SCA

Purpose: Thioglycollate broth:_____________________________________________________

Nutrient broth: _________________________________________________________
TSI: __________________________________________________________________
SIM: _________________________________________________________________
SCA: _________________________________________________________________

VI. STUDY GUIDE QUESTIONS:

1. Differentiate Blood agar plate from Chocolate agar plate.

2. Why should the petri dish be inverted during incubation and storage in the
refrigerator?

3. If the medium cannot be autoclaved, what are the preferred methods of

sterilization? Give at least 2 culture media that autoclaving is not advisable.
 HES 032- Microbiology and Parasitology
College of Nursing – RADL (AY 2021-2022)

Supplementary learning: Answer the following questions. (Cite your refences correctly. In-text
citations is encouraged)

1. If an organism grew at 25C, how would you determine experimentally whether the organism was a
mesophile or psychrophile?

2. How does the pH of the stomach protect humans from enteric pathogens?

3. You received three swabs from a wound discharge, and you placed them on enrichment media. You
noticed growth was distributed throughout all the media; there was abundant growth toward the
surface of the medium in one tube, whereas other tubes showed an equal distribution of growth
throughout the tubes. Explain why this happened.

4. Thioglycollate broth is commonly used for anaerobic culture. How can you tell if a prepared
thioglycollate medium is fresh?

5. What are the advantages and disadvantages of the pour plate method?

6. You are doing water analysis. After 24 hours, your pour-plate reveals the presence of Proteus-looking
colonies. What is the explanation why you need to eliminate these plates from your experimental data?

7. A description of colony morphology provides important information about an organism. What other
information should you include when describing physical growth characteristics?

8. List some reasons why growth characteristics are more useful on agar plates than on agar slants?

9. Explain the purpose of the following:

a. Blood in the blood agar medium

b. Crystal violet in the MacConkey agar medium
c. Increased salt concentration in the MSA medium
d. Lactose in the MacConkey agar
e. Sucrose in the TCBS medium

Compiled by: CJBregente, RMT, MPH, MSc.

Southwestern University PHINMA, College of Medical Technology
10. How is microbial growth directly dependent on the temperature?

11. Briefly explain why microorganisms differ in their pH requirements.

12. Supply the following.

Culture Medium Classification according to Purpose

function/use
a) Alkaline Peptone
Water
b) LJ medium
c) Selenite F
d) Cary Blair
e) Brain Heart Infusion
agar
f) XLD agar
g) Hektoen enteric agar
h) Skirrow agar
i) Thayer Martin agar
j) Blood agar (w/
Gentamicin)

Deadline: July 16, 2021 at 5PM