Pre-Laboratory Presentation

CULTURE MEDIA PREPARATION AND

STERILIZATION PROCEDURES
Presentation Outline

1. Introduction
• Culture Media
• Media Preparation
• Sterilization
2. Objectives of the
Exercise
3. Materials and Methods
4. Answer Sheet
 1. Introduction

• All microorganisms need

these essential matter to
grow:
- carbon, energy,
electrons

• We can classify
microorganisms based
on there nutritional
requirement

In general, microorganisms requires special

nutritional requirement to cultivate.
https://people.ucalgary.ca/~kmuldrew/cryo_course/cryo_chap8_1.html
 Culture Media

• A microbial culture medium is a mixture of substances that promotes and

supports the growth and differentiation of microorganisms.

• Culture media contain nutrients, energy sources, growth-promoting factors,

minerals, metals, buffer salts, and gelling agents (for solid media).

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 Culture Media
• Culture media are still the golden standard in pharmaceutical and food and
beverage industry to enumerate and detect microorganisms.

• Microbial culture media can be prepared as a liquid (broth), a solid (agar

plates), or as a semi-solid (deeps). Solid and semi-solid media contain a
solidifying agent such as agar or gelatin.
Liquid (Nutrient Broth) Semi Solid (Motility Medium) Solid (Potato Dextrose Agar)

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Culture Media

Important information on culture media labels.

a Name of the media.
b Concentration to prepare.
c Production and expiry date.
d Composition.
e Producer and distributor.

https://www.sigmaaldrich.com/PH/en/products/industrial-microbiology/microbial-culture-medi
 Culture Media

A. Based on form and consistency C. Based on purpose or application

1. Liquid 1. Enrichment media
2. Semi-solid 2. Selective media
3. Solid 3. Differential media
4. Assay medium
B. Based on composition 5. Characterization medium
1. Synthetic
2. Complex

https://www.sigmaaldrich.com/PH/en/products/industrial-microbiology/microbial-culture-medi
 Culture Media Preparation
How to prepare the needed culture media?
• if in dehydrated form, weigh g of medium needed
• if to be compounded, compute and weigh each component of a particular
medium or reagent

Computations:
1. Ratio and proportion
2. Percent by weight
3. Hydrated vs. non-hydrated
- get first the molecular weight, then ratio and proportion
4. Molarity
5. Normality

https://www.sigmaaldrich.com/PH/en/products/industrial-microbiology/microbial-culture-medi
Culture Media Preparation

If Synthetic Complex

using the same ratio and proportion

formula, compute for individual
component according to the total volume
of culture media to be prepared
Ratio and Proportion
Culture Media Preparation
 Sterilization
•Sterilization is the complete removal of microorganisms from an object or surfaces. Sterilization is
obtained when microorganisms are subjected to antimicrobial agents for sufficient time and at
optimum conditions.
Example: Mag sterilize ko ug culture media sa akong
Definition of TERMS! Words associated with “Sterilization” but do have different meaning!
Disinfection
-It refers to the destruction of all pathogens or organisms which can cause infection but not necessarily
spores. All organisms may not be killed but the number is reduced to a level that no longer harmful to
health
Example: Mag disinfect ko sa akong working station gamit ang 10 % Hypochlorite solution!
Mag disinfect ko sa akong kamot gamit and 70% isopropyl alcohol!
Decontamination
- the process of removing or neutralizing contaminants that have accumulated on personnel and
equipment
Example: Akong I- decontaminate akong mga glassswares sa pressure cooker!
Sanitation
the process of keeping places clean and healthy, especially by providing a sewage system and a clean
water supply
Example: Pre! Magsanitize ta sa atong classroom, mag dispose ta sa atong mga hugaw!
https://microbenotes.com/physical-methods-of-sterilization/
Sterilization

https://thebiologynotes.com/sterilization-physical-and-chemical-methods/
 Autoclave Sterilization
• The basic principle of steam sterilization, as accomplished in an autoclave,
is to expose each item to direct steam contact at the required temperature
and pressure for the specified time. Thus, there are four parameters of
steam sterilization: steam, pressure, temperature, and time.
• Also known as Steam Heat Sterilization

10 to 30 minutes
running time

https://thebiologynotes.com/sterilization-physical-and-chemical-methods/
 Ultraviolet Radiation Sterilization
Ultraviolet light inactivates microorganisms by forming pyrimidine dimers in RNA and DNA, which
can interfere with transcription and replication (Goosen and Moolenaar, 2008; Cutler and
Zimmerman, 2011)

https://doi.org/10.1371/journal.pone.0236199/
2. Objectives of the Exercise
Expected Learning Outcomes

At the end of the exercise, the student must be able to:

∙ explain the importance of culture media in microbial cultivation

∙ demonstrate proficiency in the preparation of basic culture
media
∙ elaborate the role of sterilization in microbial control and
asepsis
∙ implement a sterilization method appropriate for the intended
microbiological analysis
3. Materials and Method
3. Materials and Method(s)
 3.1. Media Preparation
1. A. Preparation of Compounded Nutrients Agar
• Prepare NA (compounded method) to be dispensed later in six test tubes (at 8 ml per tube) for eventual use
as agar slants after sterilization.

Weigh all dispense in test

cook in boiling tubes, add Sterilize (115
components add required
water bath or cotton plug (6 psi, 121°C, 15
and place in water and mix test tubes, 8 ml minutes)
microwave
beaker each)

1. B. Preparation of Dehydrated Nutrients Broth

• Prepare 50 ml of Nutrient Broth (using dehydrated medium), adjusted to pH 7.0, buffered, and dispensed
in a 125-m1 flask.

determine
Weigh transfer broth to Sterilize (115
add required initial pH and
dehydrated 125 mL E-flask psi, 121°C, 15
water and mix adjust to pH 7 if with cotton plug minutes)
Nutrient broth
needed
a. Using a clean pipet, gradually add a base (e.g. 1N NaOH) or an acid (e.g. 1N HCI) until the desired pH is reached.
b. Add a buffer (e.g. 1M KH2PO4, 10 ml/L) to prevent/reduce pH changes during sterilization .
 3.1. Media Preparation
2. Eosin Methylene Blue Agar (EMBA)
• Prepare 200 ml of EMBA (dehydrated) in a 500-ml flask for eventual use in plating
g. The agar
Compute and medium is
cook in boiling cooked when Sterilize (115
Weigh add required
water bath or everything has psi, 121°C, 15
dehydrated water and mix been dissolved
microwave minutes)
EMBA and it appears
homogenous.
 3.1. Media Preparation
3. Supplementing CM with Antibiotics
• Prepare 200 ml of EMBA (dehydrated) in a 500-ml flask for eventual use in plating

A. Antibacterial compound (Streptomycin/ Ampicillin) B. Antifungal compound Nystatin

 3.2. Sterilization
3.2.1. Sterilization Using Heat
A. Moist Heat (Steam Under Pressure): Using an Autoclave or Pressure Cooker (For culture media,
glassware, etc.)

Record observations (Daily)

5 minutes exposure

Incubate for 48 hours

to Steam Sterilization
condition
50 ml NA

Document
15 minutes exposure
to Steam Sterilization
condition
50 ml NA

Control, No exposure
to Heat and pressure

50 ml NA
 3.2. Sterilization
3.2.1. Sterilization Using Heat
B. Moist Heat (Steam Under Pressure): Using an Autoclave or Pressure Cooker (For culture media,
glassware, etc.)
 3.2. Sterilization
3.2.2. Sterilization Using using membrane filtration (For heat-sensitive substances)
Prepare Antibiotics
Solution
 3.2. Sterilization
3.2.3. Sterilization using radiation: Using UV light (For surface sterilization of work areas, etc.)

5 minutes S. marcescens
exposure
37C, 48 hrs

15 minutes S. marcescens
exposure
37C, 48 hrs