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Microbiology Lab 1
Lab Practical 1
| Question | Answer |
|---|---|
| Why are agar plates stored upside down? | Sterility - prevent water condensation from the lid dropping onto the plate Contamination Prevention - Requires bacteria to go through agar to get to bottom of the plate |
| Three types of media | agar (TSA) - solid by agar to isolate samples Broth - no solidifying agent to allow fast, expansive growth semi-solid - qualities of both agar and broth |
| What is in TSA plate | agar, nutrients, salt, and pH buffers and tags |
| What is the streak method? | dividing a TSA plate into three sections to isolate cultures |
| Pellicle Growth Pattern in Broth | grows at the top |
| Sediment Growth Pattern in Broth | grows at the bottom |
| Turbidity Growth Pattern in Broth | grows throughout entire broth aggressively; ocular density is high |
| Flocculent Growth Pattern in Broth | grows randomly throughout broth |
| Filiform Growth Pattern on a Slant | straight line in the middle |
| Arborescent Growth Pattern on a Slant | Tree-like with thick center branch |
| Beaded Growth Pattern on a Slant | Beaded down the middle |
| Effuse Growth Pattern on a Slant | Spread out growth from the center |
| Rhizoid Growth Pattern on a Slant | Branch off from thin center line |
| Echinulate Growth Pattern on a Slant | Spiny from thick center line |
| Dilution Factor | (amount of sample)/(amount of sample + amount in tube) |
| Total Dilution Factor | prior dilution factor x dilution of current container |
| Standard CFU Formula | (colony count on plate)/(total dilution factor x amount of sample plated) |
| Range of Colony Count | 30-300 |
| PCA | Plate Count Agar - provides total bacterial count |
| EMB | Eosin Methylene Blue - purple agar plate inhibits gram + bacterial growth that differentiates lactose fermenters from non-fermenters |
| CNA | Columbia Naladixic Acid - red agar plater that inhibits gram - bacterial growth that differentiates hemolytic activity |
| Differential Stain | Uses multiple dyes to help differentiate media |
| When are Darkfield Microscopy used? | For wet mounts under 10x and 40x magnification |
| When are Phase-Contrast Microscopy used? | Used for wet mounts mostly and to determine size/shape of colony |
| Simple Stain | Specimen on a slide with no more than one dye to stain, i.e. crystal violet |
| Three microscope condenser types | Brightfield, Phase-Contrast, Darkfield |
| Resulting color of a gram + bacteria | violet, because cell wall is thick and the vast amount of peptidoglycan holds in the crystal violet dye. Example: Bacillus Subtillus |
| Resulting color of a gram - bacteria | red/pink because the cell wall is thin and does not hold the crystal violet dye after the decolorizer step where acetone-alcohol is used, because the lipopolysaccharide layer is dissolved releasing the dye. Example: E. Coli |
| Which dye is used to reinforce crystal violet | Mordant's Reagent = Iodine |
| Reasons for a bad gram stain | Some bacteria are gram variable, but most will lean gram +; decolorizing agent was used for too long; culture is too old |
| Bacteria Counting Methods | Standard/Viable Plate Count - use a Quebec Plate Counter of LIVE bacteria. Accuracy increases with multiple plating. Spectrophotometeric (Turbidimetric) Analysis - use a spectrophotometer to measure optical density of both live and dead. |
| Spectrophotometer (How it measures) | check optical density via transmission of light through the sample. The larger the sample, the greater the optical density because less light passes through |
| Chromogenesis | color of the colony |
| Measurements of Colony Morphology (Types) | Whole shape, size, edge-margin, chromogenesis, opacity, elevation, surface, texture |
| Types of Colony Shape | round, irregular, filamentous (branched from center into a circle), rhizoid (random branches), curled |
| Types of Colony Edges | entire (unbroked/consistent), filamentous, undulate (patterned lumps/waves on edge), lobate (random sized lobes) |
| Types of Colony Elevation | raised, flat, convex, umbonate, growth into medium |
| Punctiform | Very small colony size (<1mm) |
| Catalase (Enzyme purpose) | Degrades H2O2 within the cell into O2 and H20 (test will have bubbles if positive). Enzyme is usually seen in gram +, for example staphylococcus |
| Catalase Test | tests for the enzyme catalase by adding H202 to a sample to see if it bubbles, which is a positive result because O2 is being released by catalase |
| Oxidase Test | Differentiates between Pseudomonadaceae (+ result, blue/purple) and Enterobacteriaceae (- result). Helps identify species with O2 as the final electron receptor because the redox reagent (dimethyl or tetramethyl-p-phenylenediamine dihydrochloride) |
| Restriction with Oxidase Test | must be read in under 30 seconds, because over time will turn positive by reacting with O2 in the air |
| Mannitol Salt Test | tests for salt resistance and mannitol sugar use. Phenol red determines sugar usage with acid as the by product. The acid lowers the pH causing a yellow color for + results. If the colony grows on the plate, then it is halophilic as well. |
| Halophillic | requires salt/tolerates salt in the environment |
| DNAse Test | Plate with DNA and methyl green indicator in it to test for presence of deoxyribonuclease (an exoenzyme) that breaks down DNA. If a DNAse enzyme is present, a yellow zone will appear around the growth area indicating + |
| Tests for Carbohydrate Utilization | phenol red sugar broth (lactose, sucrose, glucose/dextrose, mannitol) or sugar discs on a plate |
| Phenol Red Broth Test | Sugar broth with a phenol red indicator that will turn yellow (+ result) in the presence of carbohydrate utilization with an acid byproduct. A durham tube is used to check for CO2 utilization |
| Hanging Drop Mount (Purpose) | Check for motile specimens, i.e. flagella, etc. |
| Atrichous | no flagella/non-motile |
| Brownian Movement | movement caused by the bouncing of bacteria by the movement of the fluid they are suspended within |
| Run and Tumble (Specific/Difference) | run is when the motile bacteria moves towards the nutrient source and tumble is when the bacteria moves/rolls away from the nutrient source/threat |
| Monotrichous | one flagella |
| Peritrichous | flagella all around |
Created by:
drubin676
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