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
Classify bacteria as catalase negative or positive and explain your reasoning.
Catalase is an enzyme that breaks down hydrogen peroxide
Hydrogen peroxide is a toxic by-product of cellular respiration from the oxygen used as the terminal electron acceptor
H2O2 ->(catalase) 2 H2O2 + O2
What do you add to the bacteria to test for production of catalase?
Hydrogen peroxide
What does a positive result look like?
Bubbles produced
What are the bubbles?
Oxygen gas
Be able to identify the appropriate differential medium used to test for gelatinase. Identify gelatinase positive and negative, using their hallmark characteristics and explain your reasoning.
Gelatinase
Positive test: Gelatin liquifies
Negative: remains solid
Be able to identify the appropriate differential medium used to test for urease. Identify urease positive and negative, using their hallmark characteristics and explain your reasoning.
Urease
Positive test: Fuchsia/pink color
Be able to identify the appropriate differential medium used to test for sulfur reduction. Identify sulfate reduction positive and negative bacteria, using their hallmark characteristics and explain your reasoning.
What product of sulfur reduction is being detected? Hydrogen sulfide(H2S)
Negative: clear
Positive: turns black
Be able to identify the appropriate differential medium used to test for indole production. Identify indole production positive and negative bacteria, using their hallmark characteristics and explain your reasoning.
Some bacteria can breakdown tryptophan as an energy source
Tryptophanase
Tryptophan 🡪 Pyruvate + NH3 + Indole
Indole reacts with Kovac’s reagent to give a red color at the surface of the medium
Be able to identify the appropriate differential medium used to test for motility. Identify motile and non-motile bacteria using their hallmark characteristics and explain your reasoning.
Motility: Cloudy(positive), Growth staying in middle (negative)
Be able to design and interpret a dichotomous key for identification of an unknown bacterium.
Look at unknown project
Measure Zone of Inhibition on a Mueller-Hinton plate and use a table to determine antimicrobial susceptibility with the Kirby-Bauer method.
Using zone of inhibition measurements and a data table, classify antibiotic susceptibility (susceptible, intermediate, resistant) using the Kirby Bauer assay
Measure diameter of the clearing around the disc in mm.
Look at chart to determine if the organism is
Resistant
Intermediate
Susceptible
Understand how CRISPR-Cas9 can be used to edit genomes.
Interpret the results of the blue-white screen used in our CRISPR experiment.
Cas9 is a bacterial endonuclease that cuts DNA at a specific site within a larger recognition site.
The recognition sequence is a 20 bp sequence called the protospacer and is determined by the guide RNA.
The sgRNA forms a complex with Cas9 and directs the enzyme to cut at a specific sequence.
The Cas9-sgRNA complex binds to the target DNA at the PAM sequence (5’-NGG).
Does the sgRNA bind to the PAM?
The sgRNA binds to the target sequence if a match is found.
Where does Cas9 cut relative to the protospacer sequence?
Cas9 makes a ds break in the DNA 3 bp upstream of the PAM.
Double strand breaks are lethal unless repaired
Identify positive and negative agglutination reactions and correctly identify blood type for ABO and Rh factor.
ABO blood typing: the presence or absence of A antigen and/or B antigen on the surface of RBCs.
Our bodies make antibodies against the antigens our cells do not have.
Describe the applications of an ELISA.
Enzyme-Linked ImmunoSorbant Assay (ELISA)
ELISAs use antibodies to detect proteins in a patient’s sample.
Many diseases and conditions can be detected and diagnosed with ELISAs, including:
Influenza
HIV
SARS
Zika virus
West Nile virus
Lyme Disease
Researchers are also developing ELISAs that can be used to diagnose current or past coronavirus (SARS-CoV-2) infection.
Understand the steps in the ELISA procedure.
- Add the purified antigen to all the wells. incubate for 5 mins then rinse
- Add serum antibodies (samples) to the appropriate wells. Incubate for 5 mins then rinse
- Add the enzyme-linked antibody to all wells. Incubate for 5 mins then rinse
- Add enzyme substrate to all wells. Incubate for 5 mins.
Interpret the results of an ELISA experiment.
Antigen — in an antigen detection ELISA, the patient sample is tested for the presence of antigens from viruses, bacteria, etc.
Primary antibody — binds to the antigen
can be produced in a lab by injecting the target antigen into an animal and then harvesting the serum
Secondary antibody — binds to the constant region of the primary antibody
made by injecting the primary antibodies from one animal into a different animal
secondary antibodies are attached to an enzyme which catalyzes a color change when substrate is added
Substrate — changes color in the presence of the enzyme, indicating a positive result
