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*What disinfectant do we use in the lab?
T/F BSL-1 agents are known to cause disease in healthy humans.
How should petri plates with bacterial cultures be incubated? (What position?)
Inverted (upside down)
Give one example of BSL-1 agent:
GIve one example of a BSL-2 agent:
Salmonella, HIV, Lyme Disease (Borrelia Borgderferi)
Give one example of a BSL-3 agent:
Mycobacterium TB, BAcillus anthracis, Chlamydophila psittici
Give one example of a BSL-4 agent:
Samllpox, Ebola virus
What is the significance of a gram stain?
- Bacterial Identification
- Antimicrobial selection
- May provide treatment information
- Clinical evaluation of disease
*Gram Stain Procedure
- 1. Crystal Violet/ Wash (Gram + Stain)
- 2. Iodine/ Wash (Mordant)
- 3. Alcohol/ Wash (Decolorizer- removes cv from gram -))
- 4. Safranin/ Wash (Stains gram -, counterstain)
What is the most important step of the gram stain?
Decolorizing (for distinguishing the gram- from gram +)
*What are some complications with gram stains the are possible?
- Poor Prep. (over-heating, too thick a smear)
- Poor washing
- Old Sample
- Decolorize too long
What are the three shapes of bacteria?
Cocci, Rods and Spiral
What would describe cocci in chains?
What would describe cocci in clumps?
*What is the reagent for the KOH test?
3% Potassium Hydroxide
What is the purpose of the KOH test?
To differentiate gram + from gram -
What is a positive KOH test?
When the liquid becomes viscous from the lysis of a gram - cell wall liberating nucleic acid
What is a streak plate useful for?
It is good for isolating bacteria
What is a pour plate useful for?
It is more for quantification and accuracy
What is a spread plate useful for?
It is good for quantification. It is more convenient than pour and there is less contamination.
What two plate methods are good for quantification?
Pour and Spread
T/F Phase contrast microscopes can show living cells
True- and bright field as well
*T/F Oil immersion lens helps to increase focus?
T/F Enriched Nonselective media does not contain inhibitors.
List a few Enriched Nonselective Media:
- Blood agar (also differential)
- Trypticase soy agar or broth
- Mueller-Hinton agar- Anti-microbial inhibiiton assay
- Thioglycollate broth
- Sabouraud Dextrose agar- isolates fungi
Sabouraud's dextrose is best for:
Mueller-Hinton agar is best for:
Define Selective Media:
Supporting the growth of specific bacteria while inhibiting growth of others.
Examples of selective Media:
- MacConkey agar (also differential) Best for Gram - bacteria
- Eosin-Methylene blue agar (Also differential)
- Mannitol salt agar (also differential)
- Xylosine lysine deoxycholate (XLD)
- Lowenstein-Jensen medium
What is differential medium?
Contains substances like blood or acid-base indicator that only certain microorganisms will respond to in a characteristic way (ex: E. Coli on Eosin methylene blue agar= shiny green)
Name some differential mediums
- Blood agar
- Eosin-methylene blue
- Mannitol Salt
- Urease Broth
What type of blood is in blood agar?
5% Sheep blood (Can have horse and Bovine too)
What type of hemolysis do you see with alpha, beta and gamma hemolysis?
- alpha- partial
- beta- complete
- gamma- no hemolysis
Alpha hemolysin causes what type of hemolysis?
Beta hemolysin causes what type of hemolysis?
What types of inhibitors are in selective media?
Antibiotics, chemicals and dyes
What inhibits Gram + on MacConkey agar?
0.5% bile salts and crystal violet
How does MacConkey express lactose fermentation? Using what pH indicator?
Color change from light pink medium to dark red. The pH indicator is neutral red and acid production will change the color when pH falls below 6.8
Describe the colors for acid and alkaline presentation with Phenol red:
Describe the colors for acid and alkaline presentation with Neutral Red:
Catalse is present in ________________:
Most aerobic bacteria but not in obligate anaerobes.
What is the purpose of the Catalse test:
To test for the presence of the enzyme catalos which indicates either aerobic or facultative aerobic bacteria.
*What reagent is used in the catalase test? What is the reaction?
- Catalase converts 2H2O2 to 2H2O + O2
A positive catalase test will show:
What is the purpose of the oxidase test?
To detect cytochrome oxidase. Cytochrome oxidase is an enzyme used in some bacteria to convert oxygen to water molecules in the electron transport chain to ultimately generate ATP.
*What reagent is used in the oxidase test?
What will you see in a positive Oxidase test?
Filter paper will change to a blueish/purple color
Cytochrome oxidase is mainly present in:
Aerobic gram - bacteria
*How much bacteria is in a 0.5 McFarland Test?
1.2 x 10^8 CFU/ml
What is another name for the Anitmicrobial susceptibility test?
TSI stands for:
Triple sugar Iron
TSI inoculation can tell you what three things?
- Sugar fermentation
- Gas production
- H2S production
When will a pH change occur in microbial testing?
- If there is fermentation
- If amino acids are utilized
- If urea is utilized
Carboxylation will increase or decrease pH?
Increase (more basic)
Deamination will increase or decrease pH?
Decrease (more acidic)
What are the components of triple sugar iron tests?
- 0.1% Glucose
- 1% Lactose
- 1% Sucrose
- Phenol Red (acid = yellow)
- Ferrous Ammonium Sulfate (H2S production + ferrous sulfate = black)
What is the purpose of the urease test?
Urease hydrolyzes urea to ammonia and CO2. If ammonia is produced, the pH indicator phenol red will turn the urease broth red as it becomes more alkaline.
How is a TSI test recorded? Slant/Butt or Butt/Salnt?
Mannitol Salt Agar is used best for which bacteria? What is the pH indicator?
Used to grow Staphylococcus spp. and phenol red is the indicator.
What Staph species shows yellow colonies on Mannitol Salt Agar? What Staph. spp. shows white colonies?
- Staph. aureus= yellow
- Staph. epidermidis= white
What percentage of NaCL is in Mannitol Salt Agar?
In the Motility and Ornithine Decarboxylase test, what has happened if the medium turns purple?
Decarboxylation of ornithine has taken place and increased the pH (alkalinized)
Ornithine is decarboxylated into what?
- Onithine to putrescine gives purple color
Indole is formed by what process?
Trytophan to indolepyruvic acid to indole
What color is indole on the MOI test?
Red (with the use of Kovac's reagent)
What does XLD stand for?
Xylose Lysine Deoxycholate
With Eosin Methylene Blue agar, how does E. Coli look?
- Shiny metallic green whit black surrounding.
- Black= fermentation.
- Methylene blue is used to inhibit gram + growth.
- Eosin is the pH indicator.
What is the inhibitor in XLD?
What are the sugars in XLD?
xylose, lactose and sucrose
What is the pH indicator in XLD?
What amino acid is in XLD? What is it's significance?
- Decarboxylation of lysine will increase the pH and turn it dark red. Ex: Shigella spp.
What does salmonella look like on XLD?
- orange to black
- Fermentation of xylose decreases pH but decarboxylation of lysine increase pH.
- Production of H2S = black
What do E. Coli and Klebsiella look like on XLD?
There is enough sugar so that there isn't a pH change. Same color
Motility and Ornithine Decarboxylase are useful for what?
As a pH indicator, Eosin turns what color when acidified?
A red slant and yellow butt means what?
Slant and butt are yellow:
Fermentation of glucose and sucrose and/or lactose
Gas bubbles in butt and medium split implies:
If the butt shows black, there was:
If the slant and butt red throughout, it implies:
What are some general characteristics of Enterobacteriacae?
- Gram - bacilli
- Cytochrome Oxidase neg.
- Grow on MacConkey Agar
- Lactose fermenters
How many biochemical tests are there in the REMEL MICRO-ID?
*Why do we use Meuller-Hinton Agar for antibody disk diffusion?
Because it doesn't interfere with the antibacterial disks. No false +'s or -'s unlike using MacConkey or Blood Agar.