Professional Documents
Culture Documents
1. Introduction
• Culture Media
• Media Preparation
• Sterilization
2. Objectives of the
Exercise
3. Materials and Methods
4. Answer Sheet
1. Introduction
• We can classify
microorganisms based
on there nutritional
requirement
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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.
https://www.sigmaaldrich.com/PH/en/products/industrial-microbiology/microbial-culture-medi
Culture Media
https://www.sigmaaldrich.com/PH/en/products/industrial-microbiology/microbial-culture-medi
Culture Media
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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
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Culture Media Preparation
If Synthetic Complex
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
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
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