What Biosafety Level lab are we in?
Biosafety Level II lab
Lab Apparel/PPE
lab coat with top two buttons buttoned at minimum
long pants cover the whole skin area
long hair tied back. ponytail tucked in back of lab coat
safety goggles
closed toe and closed heel shoes: no sandals, clogs, mules, or crocs
Safety procedures
no food, drink, gum, or smoking
no putting on makeup
all belongings should be in the cubby (backpack, computer, etc.)
only thing at your station should be manual, pen, pencil, sharpie.
lab coat and writing utensils should be in Ziplock bag when not in use.
How-to put-on PPE
Wash hands
Put on gloves right after washing hands
Disinfect goggles with disinfectant and put them on
Put on lab coat (button at least 2 buttons)
Wipe down tabletop with disinfectant
How-to- take- off PPE
wipe down tabletop with disinfectant
disinfect gloves with disinfectant
unbutton lab coat
remove gloves and dispose of
remove lab coat
wash hands thoroughly
remove goggles and disinfect
DISPOSAL- Biohazard box
gloves only in red biohazard box (NOT in black trash can or sharp containers)
Contaminated items (paper towels used for cleaning the bench or bacteria spills need to be disposed in red biohazard box in the front
DISPOSAL- Regular trashcan
all contaminated paper towels (paper towels used to dry hands), petri dishes sleeves, cotton swab package
DISPOSAL- Biohazard bags at back
contaminated plates (cannot exceed half full, tell TA)
contaminated enteropluri test tubes
contaminated GN-ID A & B Test strips
DISPOSAL- Small bench top sharps container
contaminated razor blades, syringe needles, microscopic slides, covering slips, Kimwipes and cotton swabs
DISPOSAL- Benchtop at the back
contaminated tubes (separate liquid(left) and solid(right) tubes
transfer your tubes from the white rack into the colored racks separately
all labels need to be removed from the tubes, and disposed into biohazard box
spray the white rack with disinfectant, and put them back in order next to the wall
Micropipetting and the Basic Steps to Pipetting
measures in volume units of microliters (uL)
uses volumes between 0.5uL to 1000uL
uses disposable tips that should be discarded in sharps containers STEPS TO PIPETTING:
Select a pipette based on the volume needed
Adjust the volume on the pipette to the desired amount
Attach tip snugly
Depress plunger to first stop
Place tip in fluid and slowly release plunger, leaving the tip in the fluid a few seconds after the plunger is fully released.
Place the tip in the receiving tube, then eject the sample slowly by depressing the plunger to the second stop.
Discard the tip using the ejector into a sharp's container.
Serial Dilutions
A dilution is performed by taking a portion (called an aliquot) of one solution and adding it to an appropriate amount of liquid (diluent) in order to lower the concentration of whatever is in the solution.
A series of subsequent dilutions is called a serial dilution.
the only thing that is changing in all of these solutions is concentration.
The only difference between the stock tube and the final dilution (the tube on the far right) is how much "stuff" is in the solution.
Looking at it , this should make sense. Thesolution on the far left (called the stock solution) is a very dark pink. With each dilution, the color of the solution is considerably lighter, representing the lower concentration of each diluted solution. This is because each time a dilution is performed, a small amount of that "stuff" is transferred to the next tube.
Labeling Method
Petri dish is labelled on the bottom rather than on the lid. Write close to the edge of the bottom of the plate to preserve area to observe the bacteria culture after it has incubated.
Culture tube is labelled on the glass rather than on the cap. Write close to the bottom edge of the cap to preserve area to observe the bacteria culture. **Labels should include lab section, group initials, type of media, bacteria name, and the date.
Growth Media
refers to a liquid, or gelatin, which supports the growth of microorganisms
Liquid Media- NUTRIENT BROTH
mostly water with dissolved peptones and some beef extract. (Peptones are short chains of amino acids)
Solid Media
are prepared by adding agar to liquid medium
adding agar to nutrient broth makes nutrient agar (NA)
is necessary for the isolation of individual colonies
can be set in petri dishes (plates)
can be set in tubes (solidifies at an angle) which is referred to as a slant
agar solidifies at 40 degrees C. (can't incubate at high temps w/out agar liquifying)
Sterilization
treatment that eliminates all forms of life, including heat resistant spores and viruses
Bacterial Colonies
a collection of many bacteria (several million) on a surface such as agar.
bacterial growth on slants can also vary greatly
Different Streak patterns
a. tri- streak Isolation streak) b. lawn streak
Bacteria Morphology
Bacilli- rods Cocci- spheres Streptococci- chains of spherical cells Diplococci- pair of spherical cells Staphylococci- clusters of spherical cells
Gram Stain
Gram Positive - purple color (thick peptidoglycan Gram Negative- pink (thin peptidoglycan)
gram stain process= crystal violet-> iodine solution-> rinsed w/ethyl alcohol
RESULTS: Bacillus licheniformis: large gram positive (purple rod) Escherichia coli: small gram negative (pink rod)
Capsule Stain
Capsule: an envelope having a gelatinous consistency Procedure for visualizing the bacterial capsule- India ink (negative stain) THE BACKGROUND IS STAINED BUT NOT THE CELL
Selective vs. Differential Media
Selective- allows certain bacteria to grow but will inhibit others from growing Differential- contains substances that cause some bacteria to take on an appearance that distinguish them from other bacteria
Staphylococcus aureus
grows in mannitol-salt agar, appears yellow
Nutrient Agar
basic media used to grow our stains in our lab. (Low sensitivity and non-differential)
MacConkey's Agar (MAC)
a differential and selective medium recommended for the use in the detection and isolation of all types of enteric bacteria. (Escherichia coli, Salmonella enterica)
SELECTIVE: Gram negative bacteria
DIFFERENTIAL: Lactose fermentation
Lactose fermentation- red/pink colonies
Non-Lactose fermentation- white/clear colonies
Eosin Methylene Blue Agar (EMB)
a weakly selective and differential plating medium. (Inhibit Gram positive growth)
SELECTIVE: enteric bacteria; gram positive cannot grow -DIFFERENTIAL: lactose and or sucrose fermentation
Sugar fermentation: dark blue/black colonies; E. coli: metallic green
Non- Fermenters: white/clear/slightly pink colors
Endo Agar
is deferential and slightly selective. (Inhibit of gram positive is achieved by the incorporation of sodium sulfite and basic fuchsin)
SELECTIVE: gram negative
DIFFERENTIAL: lactose fermenters/non lactose fermenters *Lactose fermenters: clear colonies
Non lactose fermenters: red colonies
Xylose lysine deoxycholate Agar (XLD)
a highly selective medium recommended for the isolation of Salmonella enterica and Shigella
SELECTIVE: gram negative -DIFFERENTIAL: hydrogen production *Sugar fermentation: media changes to a yellow color *No Xylose fermentation: colonies appear res (shigella) *Hydrogen sulfide production: black precipitate in colonies (Salmonella enterica)
Mannitol Salt Agar (MSA)
selective for staphylococcus and is differential for staphylococcus aureus from other staphylococci.
SELECTIVE: salt tolerant organisms -DIFFERENTIAL: mannitol fermentation; changing the pH indicator
Growth on Media- organism is tolerant
Mannitol fermentation- media changes from yellow to red
Non-Mannitol fermentation- media remains red
osmotic pressure
involves 6.5% NaCl Temp: 37 degrees C
Oxidase Test
tests for the presence of cytochrome oxidase
Positive result- formation of a purple color within minutes
Negative result- no color change
Fermentation of sugars
Reversion: SUGAR-> PROTEINS-> pH CHANGE-> YELLOW-> pH CHANGE-> RED Temp: 37 degrees C
Carbohydrate Fermentation Test
objectives
principles
procedure
result and interpretation
precautions and limitations
Extracellular Enzymes
test for the presence of certain extracellular enzymes: alpha-amylase, and enzyme that catalyzes the breakdown of starch.
Hydrolysis of Starch
Temp: 37 degrees C Test for amylase using Gram's iodine solution Positive result: clear zones surrounding bacterial growth indicated the absence of starch. Negative result: a starch-iodine complex produces a deep purple to black color
Hydrolysis of Triglycerides
MA: spirit blue agar plate Temp: 25 degrees C Positive result: cleaning of the emulsion Negative result: precipitation of the blue dye
Catalase Production
positive: production of gas negative: no bubbling
Sulfur Metabolism
bacteria that are able to use sulfur will produce hydrogen sulfide or H2S.
uses media allied Klinger's Iron Agar (KIA)
Temp: 37 degrees C
Protease production
Temp: 37*C -Positive: If culture remains liquid or liquefied, gelatin hydrolysis occurred by proteases produced by the inoculums -Negative: Gelatin solidifies, then no protease was present
Ryo or KOH String Test
The alkaline solution of KOH dissolves the gram-neg. outer envelope, causing the bacteria to become sticky and tacky, thus generating a slimy emulsion. â—Ź Positive: Stringy bacteria, predicting a gram-neg. Stain result. â—Ź Negative: Non-stringy bacteria predicting a gram-positive stain result.
Alpha, Beta, and Gamma Hemolysis
Alpha hemolysis: green coloring under bacteria colonies; partial lysis of cells
Beta hemolysis: Clearing around the colonies; complete lysis of cells
Gamma Hemolysis: Growth on plate without any lytic action (non-hemolytic) Temp 37*C
Microbial Biofilms
Test the ability of bacteria to form biofilm using the dye crystal violet Temp 37*C
Motility Test Medium (MTM)
Semisolid agar media to detect motility Temp 37*C â—Ź No growth is an inconclusive result â—Ź Non-motile growth is only visible along the line of inoculation â—Ź Motile growth disperse away from the stab line of inoculation (positive result)
Antimicrobial Susceptibility Testing
An important technique to determine the effectiveness of antibacterial agents for a partial target organism is sensitivity disc assay.
This assay is also called the Kirby-Bauer method after the inventors.
The MIC is the lowest concentration of a compound that prevents growth of a particular organism. -Bactericidal: death to an organism -Bacteriostatic: block and slow the growth or metabolisms of the organism
Temp 37 degrees C