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Microbiology Lab 1

Lab Practical 1

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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