Lab practical Flashcards
Calculate total magnification.
Objective lens x ocular lens; example: 40x objective * 10x ocular = 400x total magnification
Describe bacterial cellular morphology and arrangement using the proper terminology (cocci, bacilli, spirilla, single cell/no arrangement, diplo, strepto, staphylo, tetrad, and sarcina).
Morphology: cocci(circle), bacilli(rod), spirilla (spiral)
Arrangement: single, diplo- (pairs), strepto- (chains), staphylo- (clusters), tetrad (groups of four), sarcina (groups of eight)
Identify bacterial colony morphology, including whole colony morphology, margin, elevation, pigmentation
Shape/Morphology: Round (circular), irregular, punctiform (tiny)
Margin: Entire (smooth), lobate (lobed), filamentous, undulate (wavy)
Elevation:
Flat, raised, convex, umbonate, pulvinate
Texture:
Moist, mucoid, dry
Pigmentation
Identify growth patterns in broth (uniform fine turbidity, flocculent, sediment, and pellicle
Control (normal),
uniform fine turbidity(cloudy), Pellicle(growth at the top), Sediment (bacteria sinks to the bottom), Flocculent growth (bacteria’s in bubbles formed in groups; broken up)
Successfully streak a plate for isolation
Used to obtain single colonies which area pure culture of the bacterium. Each colony arises from a CFU – colony forming unit (usually a single cell).
Be able to identify and differentiate between slides that have been prepared using Simple stain, Negative Stain, Endospore Stain, Acid Fast Stain
Gram positive: peptidoglycan and inner membrane; has a thick wall which helps it hold onto the purple dye
Gram negative: outer membrane, peptidoglycan, and inner membrane
Negative staining
Dye is acidic (acidic molecules give up H+) and carries a negative charge.
Bacterial surface is negative and repels the dye: colored background with uncolored cell.
Used to visualize bacteria that cannot withstand heat-fixing.
Also, minimizes shrinkage of cells, so size can be more accurately determined.
Acid-fast staining
Used to identify bacteria that contain mycolic acids in their cell walls
Genus Mycobacterium (tuberculosis) and to a lesser extent Nocardia are acid-fast
Mycolic acid is a waxy substance
Gives cells a higher affinity for primary stain
Gives resistance to decolonization by an acid-alcohol solution
Repels water-based stains (only weakly Gram +)
Acid-fast cells: Fuchsia/Pink
Non-acid-fast cells: Green or blue
Depends on counter-stain (brilliant green or methylene blue)
Endospore Staining
An endospore is a dormant form of a bacterium
Allows it to survive in harsh environmental conditions
Resistant to heat and chemical staining from outer covering comprised of the protein keratin
Genera Clostridium and Bacillus produce spores
Spores can be viable for many years
Viable spores have been found in 3000 years old Egyptian mummies
Spore producer:
Reddish: Vegetative/spore mother cells
Greenish: Spores
Non-Spore producer:
Reddish: All cells
*NOTE: most of our slides show endospores without the malachite green staining, so the spores will look like a clear area in the mother cell.
Simple staining
Main stains we will use:
Crystal violet
purple color
Safranin
red/pink color
Methylene blue
blue color
1.) Cover smear with stain
Use a stain tray!
2.) Rinse slide with D.I. water
3.) Blot dry with bibulous paper
Distinguish the results of Gram positive and Gram negative bacteria and be able to identify on a slide
Gram positive: purple
Gram negative: pink
Calculate the total/final dilution factor in a dilution tube following a series of dilutions.
General dilution Equation: V1D1=V2D2
V1 = Volume of first sample (or stock) added
V2 = Final volume in new tube (diluent + V1)
D1 = Dilution of first sample (=1 if stock–undiluted)
D2= Final dilution in new tube
If D1=1 (stock solution that is not diluted), then this is simplified to the Dilution Factor equation on the next slide.
Dilution factor: (Amount or volume original solution added)/ (total amount or volume made)
Example: You add 10 µL of a stock to 990 µL of water.
What is the dilution within Tube 1?
D2 = (V1D1)/V = V1/V
D2= 10.0 macroliters/ 1000.0 macroliters = 1/100 = 10^-2
Calculate the Original Cell Density of a bacterial culture (you need to know the formula OCD= CFU/v x D
Original cellular density equation: OCD = CFU/(v x D)
CFU= # of colonies on the plate
v= volume plated
D= total dilution
Example: After making a series of dilutions of a bacterial culture and plating 0.1mL each dilution the plates are incubated, and colonies are counted. One plate has 40 colonies on it. The total dilution for that plate is 1/1000 = 0.001.
What is the OCD of the starting culture?
OCD= CFU/ (v x D)
CFU= # of colonies on the plate = 40 cfu
v= volume plated = 0.1mL
D= total dilution = 0.001
OCD = 40cfu/(0.1mL)(0.001) = 400,000 CFU/mL or 4 X 10^5 CFU/mL
Understand the use of micropipettes, how to set them for a specific volume.
Micropipettes are used to transfer small volumes of liquids (0.1 µL to 1000 µL)
Micropipettes work by using disposable pipette tips, which attach to bottom of pipettor.
Tips come in different sizes based on size of pipette.
*Disposable means you want to change tips between uses!
We will be using three different pipette sizes:
Each pipette only measures a certain range!
Using a pipette for above or below that range is inaccurate and can damage the pipette!
Specific volume of pipette is written on top or side of the pipette (e.g. P-1000)
There are three positions for a micropipette:
Rest
1st Stop
2nd Stop
*1st stop for drawing in liquid
*2nd stop for ejecting liquid
The volume can be adjusted by rotating the adjustment knob or the thumb knob on top with some pipettes.
Setting the volume: When reading the volume, some pipettes have red digits to help identify the volumes.
P-20 or P-10: red digit means a decimal place (i.e 015 -> 1.5µL)
P-1000: red digit of 1 indicates you are at 1000 µL.
Identify bacteria as a lactose fermenter or a non-lactose fermenter on EMB agar and be able to explain your reasoning.
EMB agar
Which bacteria can grow on it?
Organisms that can ferment lactose. Inhibits growth of Gram positive organisms.
What does it indicate about the bacteria depending on the appearance of their growth?
Pink/purple color for normal fermentation levels
Dark purple/black with a metallic sheen for strong fermentation.
Primarily used to isolate and identify members of the Enterobacteriaceae family (Gram negative bacilli)
Identify if a bacterium is Staphylococcus aureus, another Staphylococcus species, or not a Staphylococcus species on Mannitol Salt Agar and explain your reasoning. Also, be able to distinguish between species that can ferment mannitol and those that can’t and explain your reasoning.
MSA
Which bacteria can grow on it?
Staphylococcus species
Why can’t other bacteria grow on it?
What does it indicate about the bacteria depending on the appearance of their growth?
Distinguishes between bacteria that can or cannot ferment mannitol
Color change if they can ferment mannitol?
Yellow growth
Which Staphylococcus species can ferment mannitol?
Staphylococcus aureus
Identify and interpret fermentation and gas results from Phenol Red Broth with Durham tubes and explain your reasoning.
Tests for carbohydrate fermentation
Carbohydrate fermentation is a metabolic process where a carbohydrate acts as an electron donor (to produce energy as ATP)
A single carbohydrate is added to the phenol red base to test for fermentation of that carbohydrate.
Ex. lactose, sucrose, glucose
What are the end products of fermentation that we can detect with this test?
Acid
Hydrogen gas
Carbon dioxide gas
(Alcohol)
What will we see if the bacteria can ferment the carbohydrate?
Color change: What color?
Gas production: How do we see this?
Interpret the results of the MR-VP test and explain the purpose of this test.
MR tests for mixed acid fermentation
Methyl red is a pH indicator which turns red in acidic conditions and yellow/orange in neutral/basic conditions.
VP tests for butanediol fermentation
α-naphthol followed by potassium hydroxide (KOH) is added to the culture.
The tube is mixed to oxygenate the solution.
These reagents oxidize the acetoin to diacetyl which reacts with the peptone in the medium to produce a red color.
This test is useful for differentiating between members of the Enterobacteriaceae family.
Members of the genera Escherichia, Shigella, and Salmonella are MR+/VP-.
Members of the genera Enterobacter, Klebsiella, Serratia, and Erwinia are MR-/VP+.
Classify bacteria as coagulase negative or positive and explain your reasoning.
Coagulase is an enzyme that works with normal plasma components to form protective fibrin barriers around individual or groups of cells.
Acts to shielding them from phagocytosis and other types of attack
Makes the Staphylococcus aureus more resistant to immune response and antimicrobial agents
Used to distinguish among different Staphylococcus sp.
Negative: bacteria remains in center
Positive: bacteria’s spread
