Lab Safety and Waste Disposal

Lab Safety, PPE, and Biosafety Levels

• Glass culture tubes are discarded in metal baskets at the back of the lab and tubes are separated by size
• Broken glassware and coverslips are discarded in red biohazard sharps containers
• Cotton swabs, toothpicks, and Petri plates are discarded in orange biohazard waste bags with lids
• Pipette tips and microfuge tubes are discarded in plastic biohazard waste bench containers
• Nitrile gloves are discarded in glove recycle boxes

Health Hazard Pictograms

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

Biohazard Symbol

• Biohazard symbol indicates the presence of biological hazards which can cause a risk to health

Biosafety Levels

• Level 1 is low infection severity
• Level 4 is high infection severity

Basic Microscopy Techniques

• Parts of the microscope include: arm, base, ocular lenses (10x), rotating nosepiece with four objectives (4x, 10x, 40x, and 100x), fine and coarse adjustment knobs, stage and stage controls, iris diaphragm, condenser, light source, light control/rheostat
• Resolving power is the ability to distinguish two adjacent objects from each other
• Resolution is the clarity of an image
• Ways to improve resolution are to concentrate light using a condenser (focus condenser lens), use light with a shorter wavelength, and use immersion oil
• Contrast helps distinguish from the background.
• Total Magnification = ocular magnification x objective magnification
• Working Distance is the distance between the slide and the objective lens and a smaller working distance results in a higher magnification.

Microscope Storage

• Clean each objective lens with len paper and lens cleaner
• Start with the lower-power objective (4x)
• Lower the stage all the way down
• Wrap the microscope cord
• Carefully store the microscope back in its location by holding it by the base and the arm, and place the microscope cover on top.

Eukaryotes vs Prokaryotes

• Prokaryotic cells have circular DNA, are only single-celled, and have no nucleus.
• Both prokaryotic and eukaryotic cells have DNA, can have cell walls, and have cells that function similarly.
• Eukaryotic cells have paired chromosomes, can be single-celled or multi-celled, have a nucleus, and have membrane-covered organelles.

Wet Mount

• Wet mounts allow observation of movement, unlike stains that require heat fixing.
• Slides and coverslips go in the sharps container.

Aseptic Technique

• Turbidity is the cloudiness of media indicating growth.
• Contamination is the unintended introduction of microbes such as bacteria, mold, and fungi.
• Aseptic technique should always be applied, regardless of the media to ensure there is no contamination.
• The steps for aseptic technique example using LB broth are to: flame the loop until red hot, take off cap of bacteria test tube with pinky, flame mouth of tube, dip loop, flame mouth of tube again, put cap back on, take off cap of LB broth tube to inoculate with pinky, flame mouth of tube, dip loop into broth, flame mouth of tube, put cap back on, and flame the loop until red hot again.

Gram Stain and Simple Stain

• Gram-negative bacteria have a thin peptidoglycan layer in their cell wall, stain pink or red, and an example is E. coli
• Gram-positive bacteria have a thick peptidoglycan layer in their cell wall, stain purple, and an example is S. epidermidis
• Crystal violet dyes all cells
• Iodine traps crystal violet in the thick peptidoglycan layer of gram-positive cells.
• Alcohol wash breaks the outer membrane of gram-negative cells, releasing all purple color from them.
• Safranin dyes the colorless gram-negative cells pink.
• Select either crystal violet or safranin to stain bacteria (cells will give shape and arrangement, but NOT gram).

Other Types of Stains

• Simple stains use one dye for morphological studies.
• Negative stains stain only the background and are used for capsular stains
• Structural stains stain the parts of bacteria and examples are capsule, flagella, and endospore stains
• Differential stains divide bacteria into groups and examples are gram stain and acid-fast stain

Bacterial Isolation

• Colony is a large mass of bacteria derived from a single cell
• Bacterial isolation is a technique used to separate one species from another based on morphological differences
• Streaking is a technique that dilutes bacteria to the point where single colonies are obtained
• Pure culture contains one species of bacteria
• T streak technique involves using aseptic technique, dipping the loop into the broth. Spread the loop back and forth in zone 1 of the plate, flame the loop and let it cool, starting in zone 1, continue streaking on to zone 2 by dragging the loop in a tight zig-zag finally, flame the loop, starting in zone 2, continue streaking in zone 3 by dragging the loop in a more spaced out zig-zag. Plate label with last name, first name initials, date, division ID, name of media, and name of bacteria

Enumerating Bacteria Spread Plating (Titer)

• Titer (T) = # of cells (CFU) per mL in the original sample and the calculation involves dividing the number of colonies appearing on the plate after incubation (N) by the dilution factor (DF) multiplied by the volume plated (V)
• Only calculate titer for plates with in between 20-300 colonies

Direct Microscopic Count (DMC)

• Enumeration of the total number of microbial cells in a sample using a counting chamber and a microscope
• A Petroff-Hausser Counter (thick slide that has a 0.02 mm deep chamber in the center) is used
• Advantages: quick and simple method
• Limitations: counts both living and dead cells, staining the cells is required when using a compound light microscope, counts are not precise
• Avoid going above or below its predetermined range when using a pipette to avoid affecting its internal calibration

Selective and Differential Media

• Snyder Agar is selective with a low pH of 4.5 used to select acid tolerating organisms and differentiates glucose fermenters (yellow) with non-glucose fermenters (green).
• Mannitol Salt Agar (MSA) is selective with 7.5% NaCl selects for salt tolerant organisms and differentiates mannitol fermenters (yellow, acidic) from non-mannitol fermenters (red, neutral)
• MacConkey Agar is selective with bile salts and crystal violet select for gram negative and differentiates lactose fermenters (pink, acidic) from non-lactose fermenters (yellow, basic).

Oxygen Requirements

• Fluid Thioglycollate agar and reducing agent creates oxygen gradient
• Oxygen indicator: resazurin dye (oxygen = pink ring)

Carbon Requirements

• Citrate Utilization Test uses bromothymol blue (basic) indicator to see if citrase is being utilized to convert ammonia salt into ammonia
• Stab streak technique on Simmon's Citrate Slant
• Citrase produced and converts ammonia salt into ammonia resulting in a blue color
• No citrase utilization results in no growth and a green color

Other Tests

• Methyl Red (MR) tests for acid production from glucose fermentation: sugar to lactic acid, acetic acid, or succinic acid where the results are acid formation = red and no acid is no color change
• Voges Proskauer (VP) tests for alcohol production from glucose fermentation Pyruvic acid → acetoin → EtOH where the results are alcohol formation = red ring at top of tube and no alcohol formation is no color change
• Phenol Red Lactose Broth uses phenol red to indicate lactose fermentation and a durham tube to indicate CO2 gas formation where the results are: Ferments lactose, CO2 is yellow with gas bubble. Ferments lactose: yellow, no bubble. No lactose fermentation: red, no bubble. Ammonia produced from proteins: fuschia, no bubble. If no carbon fermentation, there is peptone utilization.
• Catalase Test uses H2O2 3% to see if an organism can detoxify and use oxygen
• Majority of facultative anaerobes and aerobes use catalase to break down toxic H2O2 metabolite into harmless O2 and water where the results are catalase utilized = bubbles formed and no catalase utilized results are no bubbles
• Oxidase tests for cytochrome C oxidase using p=phenylenediamine (oxidase reagent and artificial e- donor) where the results are oxidase positive = blue and oxidase negative is no color
• Tryptophanase Test tests from tryptophanase by inoculating tryptone broth with organism and observing the color after adding Kovac's reagent where the results are tryptophanase (indole produced) = red and no tryptophanase results are no color change.
• Phenylalanine Deaminase Test tests for production of phenylalanine deaminase. Organism is stab-streaked on phenylalanine slant and Ferric Chloride 10% is added where the results are deaminase positive turns the sample green and and deaminase negative results in no color change
• Urease Test hydrolysis of urea into ammonia by urease results in urease present turning the sample pink and no urease results in no color change
• Coagulase inoculate rabbit plasma with an organism and clots. Positive where clots are present and negative when no clots = non-pathogenic
• CAMP Factor streak blood agar with S. aureus and organism to identify -hemolytic streptococci based on their formation of a substance. CAMP positive = triangle clearing and CAMP negative results where there is no clearing
• Blood Agar is differential : uses 5% sheep's blood to distinguish between Beta hemolysis results from complete red blood cell lysis, Alpha hemolysis occurs but is not a true lysis and creates green or khaki halo due to reduction of hemoglobin to methemoglobin only, and Gamma hemolysis which mean ther eis no lysis