M Bacteriology Lab
- Incubate the plates removed for
examination during the Visual Inspection
A Quality Control of Microbiological
and incubate at 35 C° for <72 hours.
Media
- Record results.
○ Satisfactory: no bacterial/fungal
Media Preparation growth
○ Unsatisfactory: bacterial/fungal
Quality Control on Culture Media growth detected
1. Sterility test
2. pH testing
A. pH testing
3. Performance testing
2
4. Specificity testing
5. Limit testing
6. Storage testing - The simplest way of testing the pH of a
culture medium is to use narrow-range pH
papers or a pH meter. pH testing can be
A. Sterility testing done during media preparation either
1
before or after autoclaving using a
well-calibrated pH meter.
- It is done by inoculating a sample of
culture media and incubating it under
appropriate condition to check microbial
growths
Visual Inspection
Figure a.1 Ingredients and their contents
A. Measuring pH of Broth
- Dip a narrow-range pH paper into a
sample of the test medium at room
temperature.
- Compare the color of the paper against the
pH color chart.
B. Measuring pH of Agar
- To test the pH of an agar medium, pour a
sample of the molten medium into a Petri
plate and let it solidify.
- Lay a narrow range pH paper on its
surface.
- Compare the color of the paper against the
pH color chart
1. For every 10-20 plates in the lot, remove 1 Adjustment of pH
plate. In most cases, adjustment of the pH won’t be
2. Perform visual inspection. needed. Minor pH adjustments can be made by
3. Record results. using
- 0.1 mol/L sodium hydroxide when the
Sterility Check medium is too acidic, and
For Sterility checking of a lot: - 0.1 mol/L hydrochloric acid when the
- In batch of 100 or fewer units: 5% of any medium is too alkaline.
lot - Use 1 mol/L (1N) sodium hydroxide to
- In larger batch: a maximum of 10 units are adjust the pH of alkaline peptone water.
tested
Use of QC Strains
M Bacteriology Lab
- Choose appropriate QC organisms based
on standards, guidelines, or
manufacturer’s instructions.
- Type culture collection organisms
○ American Type Culture Collection
(ATCC) are recommended
C Isolation of Microorganism from Air
Isolation of microorganism using settle plate
method.
2 AIR SAMPLING PROCEDURES:
1. Active
2. Passive
Figure a.2 Quality control strain, media and C. Active Sampling
expected output 1
Active monitoring a microbiological air sampler
physically draws a known volume of air through or
over a particle collection device which can be a
liquid or a solid culture media or a nitrocellulose
membrane and the quantity of microorganisms
present is measured in
- CFU (colony forming units)/m3 of air.
This system is applicable when the concentration of
Escherichia coli ATTC 25922 on EMB AGAR microorganisms is not very high, such as in an
operating theater and other hospital controlled
environments.
B Microbes in the Environment
C. Passive Sampling
2
Passive monitoring uses “settle plates”, which are
standard Petri dishes containing culture media,
which are exposed to the air for a given time in
order to collect biological particles which “sediment”
out and are then incubated.
C. Settle Plate
3
- Petri dishes containing an agar medium
are left open for a measured period of
time.
M Bacteriology Lab
- Large bacteria-carrying dust particles Figure d.3 Pseudomonas aeruginosa
settle on the medium.
- The plates are incubated and a count of
E Aseptic Technique of Transferring
the colonies is formed.
Bacteria
INTERPRETATION OF RESULTS: Colony count Inoculated: introduced bacteria into various forms
(CFU/plate) of culture media.
- The laboratory results will be available Contaminants: unwanted microbes; introduced via
within 48 – 72 hours. It is expected that 1 1. Air currents
CFU/ plate represents 1 minute of 2. Direct contact with contaminated surfaces
exposure. or your hands
- Therefore, 1 CFU/plate x 15 minutes = 15 3. Touching the media or inner surfaces of
CFU/plate. the table with any object that has not been
- A result greater than 15 CFU/plate may be sterilized
considered out of specification Asepsis: absence of microorganisms
Aseptic technique: used in microbiology to
exclude contaminants.
- All culture media are sterilized (rendered
D Colonial Morphology
free of all life)
- Sterilization is accomplished by using an
- Colony is defined as a visible mass of autoclave.
microorganisms originating from a single Agar
mother cell, - Solidifying agent
- Colony morphology can sometimes be - Extract from red marine algae
useful in bacterial identification. Broth culture
- Colonies are described based on size, - Provide a large number of bacteria in a
shape, texture, elevation, pigmentation, small space and are easily transported.
and effect on growth medium. - 0% agar
Agar Slants
- Test tubes containing solid culture media in
a small space and are easily transported.
- Like petri plates, provide a solid growth
surface , easier to store and transport than
petri plates.
- 1.5 % agar
Agar plates
- provide a solid growth surface, easier to
store and transport than petri plates
- 1.5% agar
Agar deep
- Agar is allowed to solidify in the bottom of
the test tubes
- Often used to grow bacteria that prefer
less oxygen than is present on the surface
D. Color of Colonies of the medium.
1 - Contains 0.5-0.7% agar
- Motile bacteria move away from the point
- Pseudomonas aeruginosa: Green colonies of inoculation, giving an inverted christmas
- Serratia marcescens: Red colonies tree
- Staphylococcus aureus: Golden yellow
colonies
Figure d.1 Staphylococcus aureus
Figure d.2 Escherichia coli Materials used to transfer bacteria aseptically
- Inoculating loop
M Bacteriology Lab
- Inoculating needle
For special purposes :
- Sterile cotton swabs
- Pipettes
- Glass rods
- syringes
Turbid: uniformly cloudy.
Sediments: microbial cells have on the of the tube.
Pellicle: membrane across the surface of the broth.
Flocculant: clumps of microbial cells.
Nutrient Agar Slant
- Incubate for 24 hours
- Note any pigmentation
Bacillus subtilis (white, rough colonies)
Staphylococcus citreus (lemon yellow pigment)
Pseudomonas aeruginosa
Agar deep
Motile: Christmas tree
Non-motile: straight
F Different Kinds of Streaks
Simple Steak
Overlapping Streak
Multiple Interrupted Streak
