BIOMI 2911 Practical

Bacillus Characteristics

Rods/endospores; Catalase +; Not slimy (KOH); Gram +; No oxidase; Strict aerobes/facultative anaerobes; some ferment, some respires

Enteric Bacteria Characteristics

Rods; Catalase +; Slimy KOH; Gram -; Oxidase -; Facultative anaerobes; ferments

Pseudomonas Characteristics

Rods; Catalase +; Slimy KOH; Gram -; Oxidase -; Facultative anaerobes; doesn't ferment

LAB Characteristics

Rods/cocci; Catalase -; Not slimy (KOH); Gram +; No Oxidase; Aerotolerant anaerobes; ferments

Micrococcus Characteristics

Cocci (tetrads); Catalase +; Not slimy (KOH); Gram +; No Oxidase; Strict aerobe; doesn't ferment

Staphyloccocus Characteristics

Cocci (clusters); Catalase +; Not slimy (KOH); Gram +; No oxidase; Facultative anaerobe; Ferments

Preparing a Wet Mount

Pipette 10uL water (or broth culture) onto clean slide; use loop/pick to pick up cells from edge of colony; mix cells w/ water; place coverslip on slide

Wet Mount Microscopy

Turn on and set to 10X, focus on edge of the coverslip; rotate to 40X, rotate the condenser to Ph and slide the condenser to Ph2 (click!), refocus as needed; Kohler: close luminous field, use the condenser knob to sharpen the field of view, rotate silver pins to center, open field until edges are out of frame, focus as needed; slide condenser to Ph1 (click!), oil coverslip, rotate to 100X, focus and observe

MASS-E

Motility, arrangement, size, shape, endospores

Gram Stain Procedure

Prepare 2+ clean slides and draw 2 lines with wax crayon on them; use a loop to spread cells on slides, let sit for 10 min; heat fix with pin (3x); drop 1-2 drops crystal violet over cells, let sit for 60 sec then rinse with H2O; flood with iodine (mordant), let sit for 60 sec then rinse with H2O; drop EtOH one drop at a time for 5-10 sec, immediately rinse; stain with safranin, sit for 60 sec, then rinse. Dry with bibulous paper.

Gram Stain Microscopy

Start on 10x, focus on wax; rotate condenser and lens to 40x, focus; Kohler: close luminous field, use the condenser knob to sharpen the field of view, rotate silver pins to center, open field until edges are out of frame, focus as needed; add oil and switch to 100x lens and condenser, focus

CASS-G

Color, arrangement, size, shape, gram stain results

Streak Plate Method

Label agar side of plate; sterilize loop; when cooled (10+ sec), pick up bacteria with loop and spread over a small part of the plate; flame loop and cool, draw loop into loading zone 2-4 times then streak in 2nd sector; flame loop and cool, repeat with zone 2--> 3; flame loop and cool; draw loop into 3rd sector and streak 4th sector (zig zag)

Quick Test Protocol

Place drop of x solution on a clean glass slide; with a toothpick, place a small clump of cells in the solution; mix gently; wait for results

Catalase Test

Uses hydrogen peroxide bubbles indicated catalase was present; bubbles = catalase +

KOH Test

Confirms gram stain based on whether 3% KOH can lyse bacterial cell wall and release DNA; slimy = G-, not slimy = G+

Oxidase Test

Identifies bacteria that have cytochrome oxidase; ONLY DONE ON G- BACTERIA; do on blotting paper; red = oxidase +

Selective Media

Suppress growth of unwanted bacteria and encourage growth of desired microbes

Differential Media

Allows growth of several types of microbes and displays visible differences among those microbes

EMB Agar

Eosin Y and Methylene blue inhibit G+ growth, lactose indicates pH change based on lactose fermentation (metallic color = ferments lactose); selective and differential

MacConkey Agar

Selective (bile salts and crystal violet) for G- bacteria; differential (neutral red) based on Lac fermentation- turns purple

MRS Agar

Selective (acetate, tween 80, citrate) for G+ bacteria; enriches for LAB (anaerobic incubation)

Vitamin Free Minimum Medium (VFM)

No vitamins added, very few minerals in the plate- allows robust bacteria to grow (not fastidious)
E. Coli should grow, LAB shouldn't

PEA Agar

Selective for G+ bacteria (especially cocci) and inhibits most G- bacteria

MSA Agar

Mannitol Salt Agar- Both selective and differential; selects for salt-tolerant organisms and differentiates mannitol fermenters from non-mannitol fermenters (turns yellow if it ferments)
Staph. aureus grows and ferments, Micrococcus grows but doesn't ferment

Strict Aerobe

Requires O2 because they only make ATP via respiration
Purple, growth only at top, no cracks

Strict Anaerobe

Does not use O2 to make ATP, and O2 is lethal
Color change, growth only at the bottom

Facultative Anaerobe

Prefer to respire O2, but can ferment
Color change, growth concentrated at top but throughout

Aerotolerant Anaerobe

Can only ferment, but O2 is not lethal
Color change, evenly distributed growth with top 2mm relatively clear

What does a color change in a BCP shake indicate?

Acid production during fermentation- pH drops

What do cracks in a BCP shake indicate?

Gas is produced during fermentation

Caseinase (Protease) Test

Add Casein (substrate) to the media, spot inoculate Bacillus, and incubate; degradation can be directly visualized as clearing around bacterial growth.

Amylase Test

Add Starch (substrate) to the media, spot inoculate Bacillus, and incubate; add iodine (indicator; binds to starch
forming dark brownish color) to visualize
clearing.