Practical Microbiology Flashcards
What is Aseptic Technique?
A barrier between you
/ the environment
To protect:
- You & your colleagues
- The environment
- Your experiment
-and your experiment
Class II / III Microbiological Safety Cabinets
Class II and Class III microbiological safety cabinets are not always needed, but they are used when working with hazardous materials or aerosols.
When to use a Class II cabinet
-When working with microbiological hazards, such as pathogens, dust, or spores
-When working with samples that could be contaminated by other samples or the laboratory atmosphere
-When performing regular testing where consistency and repeatability are important
When to use a Class III cabinet
-When working with the most sensitive or potentially harmful pathogens
-When working with Hazard Group 3 and Hazard Group 4 pathogens in Containment Level 3 or 4 facilities
Checklist for Aseptic Techniques
Checklist
* Work area
* PPE / Hygiene
* Materials
* Handling
* Waste Disposal
Nutritional Requirements
– All cells need access to large amounts of carbon,
nitrogen, phosphorus, sulfur, and oxygen
(macronutrients) to build macromolecules.
– Various micronutrients are also required by
microbes:
* Includes several metal ions (Na+, Mg2+, Mn2+, etc.)
* Often required for protein structure/activity
Bacterial Culture Media
- Any substance (or mixture of substances) used to grow
microorganisms in the laboratory - Microbes can be grown in the lab on both solid (agar plates)
and liquid media (broths).
Nutrient Usage
Limitation of 1 key
nutrient can dictate the
rate of growth
Catabolite Repression:
Bacteria can prioritise use
of certain carbon sources
over others
Nutritional requirements for growth
- Prototrophs can synthesize
all needed macromolecular
precursors from a single
carbon source and inorganic
molecules. - Auxotrophs require
supplementary factors for
growth (e.g. one or more
amino acids).
Complex vs Defined Media
Complex: unknown
chemical composition
Defined: precisely defined
chemical composition
Bacterial colonies
Single colonies isolated
on agar plates
1 colony indicates that 1
viable bacterial cell
(“colony forming unit”)
was present in a given
sample
Liquid culture (broth)
-Clear
-Turbid
Measuring bacterial growth
- Turbidity
(optical density) - Direct cell counts
(microscopy) - Viable counts
Live colony counts
- Turbidity
Spectrophotometer sends light through a culture
- Direct counts
Pros: Cheap, fast, easy
Cons: You can’t differentiate living vs. dead cells
- Viable counts
Plate out a sample on
e.g. nutrient agar: 1 colony will
grow from each viable (live) bacterial
cell = 1 colony forming unit
= c.f.u
1st you may need to
dilute the culture
* Dilutions are spread
plated
* After incubation, colonies
are counted.
* Colony forming units
(c.f.u) per milliliter of initial
culture is calculated
2nd Spread / Pour plate
3rd Colony Forming Units per millilitre =
c.f.u. mL-1
159 (plate count) x 103 (dilution factor) = 1.59 x 105 per 100 μl = 1.59 x 106 mL-1
Continuous culture (chemostat)
The human gut can be considered a continuous culture
* Nutrients are continually added.
* Waste and excess microbes are occasionally removed.
bacteria aren’t always present in
such high numbers
A filter apparatus can concentrate the cells.
important for testing clean
water
Obtaining PURE culture
– One of the benefits of a solid medium is that cells
are held in place on the surface and can be isolated.
– This enables separation of a mixture of cells into a
pure population.
done by spread / pour plating (Colony Isolation)
Selectivity
How do we select for particular bacteria to grow?
* Reduced O2 will select for Anaerobes
* Increased temperature will select for thermophiles
* Acidic media will select for acidophiles
* Antibiotics will select for non-susceptible bacteria
* Specialised selective / differential media
MacConkey Agar
Selective:
*contains bile salts – selective for enterics
*inhibition of Gram-positives
Differential:
*contains lactose and neutral red dye
*lactose fermenting – pink to red
Selective agar can be used to “hunt” for particular bacteria
Mannitol Salt agar
Selective:
* Contains high salt (7.5-10% salt)
* Selective for Staphylococci
Differential:
* Contains mannitol and phenol red
dye
* Mannitol fermentation – acid
byproduct converts phenol red -
yellowS. aureus = yellow
Other Staphylococci = pink
Bacterial Characterisation
- Morphology – Colony / Cell Shapes
- Function – Motility, Susceptibility, Metabolism
- Molecular – Polysaccharides / Protein / DNA
Describing bacterial cell morphology
-Cocci
-Rod
-Spirochete
-Branched rod
-Curved rod
-Filamentous
Colony Morphology
-Compact Circular Colonies
-Filamentous Colonies
Gram Positive/Negative
Cell Wall Structure: Gram-positive bacteria have a thick peptidoglycan layer, while Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane containing lipopolysaccharides.
Staining Result: Gram-positive bacteria retain the crystal violet stain and appear purple, whereas Gram-negative bacteria lose the crystal violet stain and take up the counterstain (safranin), appearing pink/red.
Clostridia and Bacillus
Gram positive rods
Endospores in the centre of sporangium = resistant to Gram staining
Mycobacterium tuberculosis
Neither gram positive or negative
Acid Fast Stain: Acridine orange
Analytical Profile Index (API)
API strips comprise 20 mini biochemical tests
Each strip is scored according to the result obtained in each well
yielding a numerical profile, which is compared to a database, giving
a most likely identity
API strips comprise 20 mini biochemical tests
Multiple systems able to ID > 600 species of bacteria
*e.g. Fermentation of glucose produces acid →yellow colour
Antibiotic sensitivity profiles
- Simultaneous testing of multiple antibiotic susceptibilities
- “Zone of inhibition” – Guidelines for effective antibiotic dose
Molecular Typing: Surface
Structures
Serotyping Uses antibodies
to detect key surface antigens – eg O-
polysaccharides
Phage typing Uses bacteriophages to detect key surface structures that phage use to infect the cell
Summary
- Bacterial physiology and growth requirements are
diverse and careful aseptic technique is needed to
avoid contamination and infection. - Differences can be exploited for isolation /
identification - Traditional methods often require the ability to
culture the organism, whereas molecular methods do
not
Genetic Profiling
Genetic Profiling
Pulsed Field Gel Electrophoresis (PFGE
