Lab Safety and Hazard Pictograms

Lab Safety, PPE and Biosafety Levels

• Discard glass culture tubes in metal baskets at the back of the lab, separating by size
• Dispose of broken glassware and coverslips in red biohazard sharps containers
• Cotton swabs, toothpicks, and petri plates go into orange biohazard waste bags with lids
• Discard pipette tips and microfuge tubes in plastic biohazard waste bench containers
• Nitrile gloves are discarded in glove recycle boxes

Health Hazard Pictograms

• Flame indicates flammable materials
• Flame Over Circle signifies substances that can cause combustion (oxidizers)
• Corrosion indicates substances causing skin corrosion, burns, and eye damage
• Health Hazard warns of carcinogens, reproductive toxicity, and organ toxicity
• Skull and Crossbones indicates acute toxicity (fatal or toxic)
• Environment signifies aquatic toxicity
• Exclamation Mark warns of skin and eye irritants and toxic or harmful substances
• Gas Cylinder indicates gas under pressure
• Exploding Bomb indicates explosives, self-reactives, and organic peroxides

Biohazard Symbol

• The biohazard symbol indicates the presence of biological hazards that pose a risk to health and it can be found on bags/containers with biological waste.

Biosafety Levels

• Level 1 is the lowest risk
• Level 4 is the highest risk

Basic Microscopy Techniques

• Key parts of a light microscope include: the arm, base, ocular lenses (10x), rotating nosepiece with four objectives (4x, 10x, 40x, 100x), fine and coarse adjustment knobs, stage and stage controls, iris diaphragm, condenser, light source, and light control/rheostat
• Resolving power distinguishes two adjacent objects from each other
• Resolution refers to the clarity of an image
• Resolution can be improved by concentrating light with a condenser lens, using shorter wavelength light, and using immersion oil
• Contrast is distinguishing an object from the background
• Total Magnification = ocular magnification x objective magnification
• Working Distance is the distance between the slide and objective lens
• A smaller working distance results in higher magnification

Microscope Storage

• Clean each objective lens with lens paper and lens cleaner
• Start with the lower-power objective (4x) and move through to the high-power objective (100x)
• Clean ocular lenses
• Turn the rotating nosepiece to the 4x objective after cleaning all lenses
• Lower the stage all the way down
• Wrap the microscope cord
• Store the microscope carefully, holding it by the base and arm, and cover it

Eukaryotes vs. Prokaryotes

• Prokaryotic cells contain circular DNA, are single-celled and lack a nucleus e.g. Bacteria
• Eukaryotic cells have paired chromosomes, can be single or multi-celled, have a nucleus and membrane covered organelles e.g. Protists, Fungi, Plants and Animals
• Both cell types contain DNA, cell walls and function similarly

Wet Mount Procedure

• Wet mounts allow observation of movement, unlike heat-fixed stains
• The green color is from chlorophyll, allowing the organism to obtain energy
• Slides and coverslips are discarded in sharps containers

Aseptic Technique

• Turbidity is the cloudiness of media indicating growth
• Contamination is the unintended introduction of microbes
• An example of aseptic technique includes flaming the loop, taking off the bacteria test tube cap with your pinky, flaming the mouth of the tube, dipping the loop and replacing the cap, then flaming the loop again
• Aseptic technique should be applied to all media, regardless of type, to ensure no contamination

Gram Stain

• Crystal violet dyes all cells
• Iodine traps crystal violet in the thick peptidoglycan layer of gram-positive cells
• Alcohol wash breaks down gram-negative cell membranes, releasing all purple color
• Safranin dyes the colorless gram-negative cells pink

Gram Stain and Simple Stain Examples

• Gram-positive bacteria (e.g., S. epidermidis) have a thick peptidoglycan layer and stain purple
• Gram-negative bacteria (e.g., E. coli) have a thin peptidoglycan layer and stain pink or red
• Simple stains use one stain, such as crystal violet or safranin, to determine shape and arrangement, but NOT gram type.

Bacterial Shape and Arrangement

• Common bacterial shapes include cocci (spherical) and bacilli (rod-shaped)
• Arrangements include coccus, diplococci, streptococci, staphylococci, tetrad, and sarcina

Other Types of Stains

• Simple stains use one dye for morphological studies
• Negative stains stain the background
• Structural stains stain parts of bacteria e.g. capsules, flagella or spores
• Differential stains divide bacteria into groups based on reactions to various dyes

Bacterial Isolation

• A colony is a large mass of bacteria derived from a single cell
• Bacterial isolation is a technique to separate one species from another based on morphological differences
• Streaking is a technique that dilutes bacteria to obtain single colonies
• A pure culture contains one species of bacteria

T-Streak Technique Steps

• Using aseptic technique, dip the loop into the broth and spread the loop back and forth in zone 1 of the plate
• Flame the loop, let it cool, then continue streaking to zone 2 in a tight zig-zag pattern
• Flame the loop again, then streak into zone 3 in a more spaced-out zig-zag pattern
• If the loop is too hot, tap the loop on zone 3 until sizzle sound stops to avoid killing the bacteria
• Plate label must include last name, first name initials, date, division ID, name of media, and name of bacteria

Enumerating Bacteria Spread Plating (Titer)

• Titer (T) is the calculated number of cells (CFU) per mL in the original sample
• N is the number of colonies appearing on the plate after incubation
• DF is the dilution factor
• V is the volume plated
• Only calculate titer for plates with between 20-300 colonies

Direct Microscopic Count (DMC)

• A direct microscopic count is used to enumerate the total number of microbial cells, both dead and living.
• The Petroff-Hausser Counter is used, which is a thick slide with a 0.02mm deep chamber in the center
• Staining is required when using a compound light microscope as counts are not precise

Pipette Usage

• Always avoid going above or below the pipette's predetermined volume range to avoid affecting the pipette’s internal calibration

Selective and Differential Media

• Selective media selects for growth of specific microorganisms
• Differential media distinguishes between microorganisms

Snyder Agar

• Snyder Agar is selective for acid-tolerating organisms with pH 4.5
• Snyder Agar differentiates based on glucose fermentation; glucose fermenters appear yellow, while non-glucose fermenters appear green.

Mannitol Salt Agar (MSA)

• MSA selects for salt-tolerant organisms using 7.5% NaCl
• MSA differentiates based on mannitol fermentation; mannitol fermenters produce a yellow, acidic result, while non-mannitol fermenters produce a red, neutral result.

MacConkey Agar

• MacConkey Agar selects for gram-negative bacteria using bile salts and crystal violet
• MacConkey Agar differentiates based on lactose fermentation; lactose fermenters produce a pink, acidic result, while non-lactose fermenters produce a yellow, basic result.

Tricks for Remembering Color Changes

• Snyder: Lemonade is sugary and yellow (yellow colonies are glucose fermenters), Kale is green and bitter (green colonies are not glucose fermenters)
• MSA: Lemonade is sugary and yellow (yellow colonies are mannitol fermenters), Dry red wine is gross (red colonies are not mannitol fermenters)
• MacConkey: Strawberry milk contains lactose (pink colonies are lactose fermenters), No lactose in lemonade (yellow colonies do not ferment lactose)

Oxygen Requirements

• Fluid Thioglycollate creates an oxygen gradient
• Resazurin dye indicates the presence of oxygen and will appear as a pink ring

Carbon Requirements

• The Citrate Utilization Test determines if citrase converts ammonia salt into ammonia
• The Simmon's Citrate Slant is used with the stab streak technique
• Citrase production results in a blue color
• No citrase utilization results in a green color

Methyl Red (MR) Test

• The Methyl Red Test tests for acid production from glucose fermentation
• Methyl red is added and 3 drops give a red result for acid formation, but no color change represents no acid

Voges-Proskauer (VP) Test

• The Voges-Proskauer (VP) Test tests for alcohol production from glucose fermentation using pyruvic acid → acetoin → EtOH
• Barrit’s A and B are added and show a red ring indicates alcohol formation

Phenol Red Lactose Broth

• Phenol Red Lactose Broth tests for lactose fermentation and CO2 gas production
• Lactose fermentation with gas production results in a yellow color with a gas bubble
• Lactose fermentation without gas production results in a yellow color with no bubble
• No lactose fermentation results in a red color with no bubble
• Ammonia production results in a fuchsia color with no bubble
• If no carbon is fermented, peptone is utilized

Catalase Test

• The Catalase Test sees if an organism can detoxify and use oxygen
• If bubbles form, catalase is utilized
• If no bubbles form, catalase is not utilized

Oxidase Test

• The Oxidase Test tests for cytochrome C oxidase, using p-phenylenediamine
• An oxidase-positive reaction is blue
• An oxidase-negative reaction is no color

Tryptophanase Test

• The Tryptophanase Test tests for tryptophanase
• Tryptophan becomes indole, pyruvic acid, and ammonia and it reacts with Kovac's reagent to produce red
• A red color indicates tryptophanase is produced and if there is no color change then it is not produced

Phenylalanine Deaminase Test

• The Phenylalanine Deaminase Test tests for production of phenylalanine deaminase (phenylalanine → phenylpyruvic acid and ammonia)
• If deaminase is positive, it turns green
• If deaminase is negative, there is no color change

Urease Test

• The Urease Test tests for hydrolysis of urea into ammonia by urease
• If Urease is present it will turn pink
• If there is no urease, there will be no color change

Coagulase Test

• The Coagulase Test inoculates rabbit plasma to see if the organism clots, converting fibrinogen to fibrin
• If clots form, coagulase is pathogen
• If no clots form, coagulase is non-pathogenic

CAMP Factor Test

• The CAMP Factor test detects B-hemolytic streptococci formation
• A triangle clearing indicates a CAMP positive test
• No clearing indicates a CAMP negative test

Blood Agar Test

• Blood Agar differentiates based on hemolysis using 5% sheep’s blood
• Beta hemolysis represents complete lysis of red blood cells
• Alpha hemolysis shows a green/khaki color halo surrounding growth, indicating reduction of hemoglobin into methemoglobin
• Gamma hemolysis represents no lysis of red blood cells