Lab practical

Objective lens x ocular lens; example: 40x objective * 10x ocular = 400x total magnification

Morphology: cocci(circle), bacilli(rod), spirilla (spiral) Arrangement: single, diplo- (pairs), strepto- (chains), staphylo- (clusters), tetrad (groups of four), sarcina (groups of eight)

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

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)

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).

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

Gram positive: purple Gram negative: pink

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

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

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.

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)

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

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?

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+.

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

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

Gelatinase Positive test: Gelatin liquifies Negative: remains solid

Urease Positive test: Fuchsia/pink color

What product of sulfur reduction is being detected? Hydrogen sulfide(H2S) Negative: clear Positive: turns black

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

Motility: Cloudy(positive), Growth staying in middle (negative)

Look at unknown project

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

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