LAB Test 2 Flashcards
Ex. 9-16
Form for scientific notation
a × 10b
1 < a < 10
b gives the order of magnitude
What are plate counts used for?
Determining water, milk, food quality
103
kilo
100
Base unit
10-3
milli
10-6
micro
10-9
nano
Why are serial dilutions used?
Method used to efficiently calculate the number of microbes in a sample.
Dilutions will thin out the lawn until we can see single colonies, which we can physically count
Diluent
a fluid used to dilute the concentrated sample
(e.g. water)
Aliquot
a smaller volume withdrawn from a total sample volume
Dilution factor
Fraction by which your original sample concentration is diluted
Equation for dilution factor
DF = Vf / Vi
Vf = aliquot volume + diluent volume
Equation for dilution
Dilution A = (total dilution)(original concentration)
Equation for final dilution
Final dilution = product of total dilutions at every step in the series
Final sets of the tubes have the highest ______ and the lowest ______.
dilution; concentration
P1000
200-1000µL
0 | 9 | 0 (=900µL)
Larger blue tips
P200
20-200µL
1 | 0 | 0 (=100µL)
Smaller yellow tips
Using micropipettors
- Depress plunger to FIRST STOP befor you place it into the sample
- Slowly return the plunger to the up position to suck up the liquid
- Release liquid by depressing plunger all the way to the SECOND STOP
- Change tips after every transfer
Spread Plating
- Dip the spreader into alcohol; let excess alcohol drip back into the jar before flaming (to sterilize)
- Wait until the plate spreader is cool
- Apply liquid culture to plate and spread
- Take only 100 µL from the tube to spread on the plate
- This will result in a 1/10 dilution
- Take only 100 µL from the tube to spread on the plate
When are plates considered countable?
30-300 colonies
TNTC (too numerous to count): >300 colonies
TFTC (too few to count): < 30 colonies
Calculating the number of bacteria in the original sample
O.T. Concentration = Number of CFUs / (Total Dilution)(Volume Plated)
O.T. Concentration = (Number of CFUs)(Total Dilution Factor) / Volume Plated
units: CFU/mL
Growth Characteristic Tests
- Osmotic pressure
- O2 requirements
- pH
- Temperature
- Pigment production
Strict aerobe
connot exist without 02
Strict anaerobe
cannot live in the presence of O2
Facultative anaerobe
can grow with or without O2, but they grow better with O2 present
Aerotolerant anaerobe
cannot use O2 for growth, but can tolerate it
Microaerophile
Grow best in high CO2 / low O2 environment
Thioglycolate medium
- used in culturing anaerobes
- sodium thioglycolate binds free O2
- Creates an O2 gradient (most O2 at the top of the tube and progressively less O2 towards the bottom)
- agar reduces the rate of diffusion of O2 into and out of medium
- Contains resazurin, an indicator dye that turns pink in the presence of O2 and is colorless in absence of O2
Examples of strict (obligate) aerobes
- Bacillus subtilis*
- Mycobacterium tuberculosis*
- Mycobacterium leprae*
Examples of strict (obligate) anaerobes
- Clostridium perfringens*
- Clostridium botulinum*
- Clostridium tetani*
Examples of Facultative Anaerobes
- Staphylococcus spp.*
- Corynebacterium spp.*
- Bacillus anthracis*
- Klebsiella spp.*
Examples of Aerotolerant Anaerobes
Clostridium sporogenes
Anaerobic jar
- used to incubate regular media in an anaerobic environment
- Uses catalysts to combine O2 with H2 to form H2O
- packet - gives off CO2 after about 3 hours to create CO2 atmosphere in the absence of O2
- Contains methylene blue indicator strip
- Blue= O2 present
- White = O2 absent
What does temperature affect in bacteria?
function of enzymes and proteins essential for growth and survival
Maximum growth temperature
highest temperature in which species will grow (will usually have minimal growth rate)
Minimum growth temperature
lowest temperature in which species will grow (will usually have minimal growth rate)
Optimum growth temperature
Temperature at which species have the highest growth rate
Mesophile
Grows best between 25-40º C
(e.g. human gut flora)
Psychrophile
Grows between 0-5ºC
(e.g. bacteria in the deep sea, Arctic regions; food spoilage microbes)
Thermophile
Grows between 45-65ºC
(e.g. bacteria in hot springs; hyperthermophiles can survive near-boiling conditions like deep sea vents)
What controls pigment production?
enzyme activity
pigment is the product of biochemical pathways involving enzymes
some bacteria make pigments in certain conditions (usually temperature)
Diffusible pigment
a pigment that can diffuse into agar from the colonies (soluble in water)
Non-diffusible pigment
a pigment that is confined to the colony
Pseudomonas aeruginosa
- opportunistic and nosocomial pathogen
- produces two types of soluble pigments
- fluorescent green
- blue
- Hallmark of infection: “blue pus”
Osmosis
a movement of water across a semi-permeable membrane from a lower solute concentration to a higher solute concentration
Osmotic pressure
pressure that must be applied to the solution to equalize the flow of water across the semi-permeable membrane in both directions
Hypertonic environment
solute concentration outside the cell is higher
water flows out of the cell
Plasmolysis
cytoplasm shrinks; cell collapses
result of hypertonic environment
Hypotonic environment
Solute concentration outsie the cell is lower
water flows into the cell
Cell lysis
cytoplasm swells; cell explodes
result of hypotonic environment
Isotonic environment
solute concentrations across the membrane are equal
no water flow in either direction
no osmotic pressure
What will happen if there is improper pH?
“fold” the proteins differently, impairing their function
What pH do most bacteria grow at?
neutral pH (6.5-7.5)
some grow at acidic pH (low) or alkaline pH (high)
Acidophiles
prefer acidic conditions (low pH)
EX: Thiobacillus ferrooxidans lives in coal mines and can survive at pH 1
Alkaline bacteria
prefer alkaline conditions (high pH)
EX: Vibrio cholerae can live up to pH 9
Endospores
- encapsulate genetic material (DNA) and essential cell components
- Enable bacteria to survive unfavorable environmental conditions
- starvation
- Extreme heat/cold
- Dessication
- Toxic chemicals/antibiotics/radiation
- Once favorable conditions return, the endospores can germinate to form vegetative cells
Endospore staining
Primary stain: Malachite green (10-15 min)
Counterstain: Safranin (30 sec)
Vegetative cells: pink
Endospores: blue
Transient bacteria
contaminants; may be present for a finite time
may be pathogenic
Resident bacteria
permanent residents of normal human flora
normally, not pathogenic
Ignác Semmelweis
introduced concept of hand-washing
suggested washing with chlorinated water
Common skin microbiota
- Corynebacterium diphtheriae*
- Staphylococcus aureus*
- Micrococcus luteus*
- Staphylococcus epidermidis*
- Pityrosporum ovale*
Isolation by dilution
dilute the culture enough that single cells give rise to single colonies on a plate (all cells in a single colony are clones)
Pour plate, Spread plate, Streak plate
Colony morphology
- color
- white, cream, tan, brown, black, purple, yellow, red, etc
- size
- pinpoint, small, medium, large
- shape
- raised, flat, convex, fried egg, dimpled (caved in), growth in agar
- margin
- smooth, irregular, filamentous, rhizoid, wavy/swarming, entire, filamentous, spreading
- consistency
- shiny, glistening, dry, powdery, wrinkled, rough, dull/matte, mucoid
T-Streak Plate
- Pick up culture
- Inoculate in Zone 1
- Flame loop
- Streak through Zone 1 into Zone 2
- Flame loop
- Streak through Zone 2 into Zone 3
Cotton Applicator Stick
- Sterile polyester-tipped swab
- Swab the surface of interest and place it in the provided case
- Case = glass ampule which contains general purpose medium
- survives for up to 72 hours
Why is quality control used?
to ensure that:
- procedures and results can be evaluated for accuracy
- there are controls provided for comparison
- the dyes/reagents are in good condition
ALWAYS use when identifying an unknown organism with Gram staining
Gram staining
- Differentiates two large groups of bacteria based on their cell wall compositon
- Gram negative (Gm-) [pink] and Gram positive (Gm+) [purple]
- helps to determine appropriate antibiotic treatments
- Gram negative (Gm-) [pink] and Gram positive (Gm+) [purple]
Primary stain: crystal violet (1 min)
Mordant: Gram’s iodine (1 min) -complexes crystal violet to peptidoglycan layer
Decolorizing agent: ethanol (10-20 sec) - dissolves lipids in Gm-cells enhancing decoloization and dehydrates Gm+ cells closing off pores
Counter-stain: safranin (1 min)
Bacterial cell wall
- composed of peptidoglycan
- polymer of amino acids (“peptido-“) and sugars (“-glycan”)
- layered on top of the plasma membrane
- Thickness of the layer of peptidoglycan comprising the cell wall determines Gram character
- Thick = Gm+
- Thin = Gm-
What is the charge of the exterior of the cell?
anionic charge
What type of dye is crystal violet?
cationic dye
What will happen if you forget to add mordant?
Gm+ may appear pink
What will happen if you decolorize too long?
Gm+ may appear pink
Acid-Fast Stain
Used to identify Mycobacterium spp. on the basis of their mycolic acid layers
Primary dye: Kinyon’s TB carbolfuchsin (KF) - chemically binds to mycolic acid in cell wall
Decolorizer: Acid alcohol - washes away KF if not bound to mycolic acid
Counterstain: Methylene blue
Interpretation: dark red/pink rods (positive); blue (negative)
Glycocalyx
slimy layer surrouding cells made of polysaccharides (“sugar coat”) or sometimes of proteinaceous material
protects cell from immune system
Virulence factor
any bacterial adaptation that makes them more pathogenic
- Capsule
- Nuclear matter
- Cytoplasm
- Cell membrane
- Cell wall
- Pili
- Flagellum
Capsule staining
The capsule repels dyes so it cannot be stained directly
Negative Staining
- Background is stained
- Capsule is visualized as a halo around the cell
- Indirect visualization
