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- Heat 172C 2 hours
- - all of the water will evaporate if boiled
- - so dry heat is best
121C,15 minutes, 15 PSI
Why do we need a media?
A nutrient food to help it reproduce and multiply
What does sterilization mean?
Process of getting rid of all forms of life and viruses
At what temperature will nutrients not survive?
-Cabinets are where you place the dishes, so best place to put UV light to decontaminate
-It cannot penetrate through the container, so UV is not strong
Enthylene Oxide (gas)
- -Used in industry or in hospital
- -It penetrates through materials
It doesn't kill everything, it is partial sterilization.
-Vegetative cell (spores)
- -If the environment is not good it will stop producing but it will survive but eventually die
- - They sporulate, hibernate
- - slows down metabolism and use remaining energy to create a shield, barrier
Media (Physical Properties)
- 1.5% Agar
- ~add to 100 ml liquid
Semi-solid agar (Media)
- 0.4-0.8% Agar
- to dissolve agar, it has to be at 100 C
- at 42-45 C it will start solidifying and dissolve
Give all nutrient, amino acid and sugar
Defined synthetic medium
A medium in which specific nutritional requirements or chemical compositions needed for microbial growth
Complete (rich) medium
Gives everything, it has all the nutrients
ass something or take away that some organism will grow or don't
-a medium which is used to differentiate different types of microorganisms based on their different colours or colony shapes.
-Differential media or indicator media distinguish one microorganism type from another growing on the same media
- Used for wet mount- not for stained prep
- Great for spores/capsules
Phase contrast microscopy allows the viewing of unstained specimens
Refractive index 1.2
Use Immersion oil on the slide it will
The function of immersion oil is to direct the light from the microscope directly to the slide and stop it from refracting.
Thick peptidoglycan layer
Thin peptidoglycan layer
What is the purpose of gram staining?
- Staining gives you visualization of the cell
- Direct staining and Negative staining
staining of the organism and cells
Staining the background
-stain on all the cells
-Simple stains highlight an entire microorganism so that cellular shapes and basic structures are visible. Simple stains commonly used include methylene blue, carbolfuchsin, crystal violet, and safranin.
- stain that are color
- positively charge will only stain
It allows you to differentiate the different groups or individual around it
Spore staining (Specific)
Spore staining is used to identify bacteria that have endospores, or hard outer shells that do not absorb dye. Spores are heated to break the shells, then malachite green dye is applied to dye the spores.
A primary stain is a technique in which certain types of dyes are used to stain tissue and bacteria so that they become easily visible under the microscope.
It stains the outer membrane
- removes excess primary dye and/or removes the primary dye and outer
- membrane if it is unable to attach to the cell (is negative)
attaches to the inner membrane in the event that the primary stain doesn't (is negative)
Mordant (means biting, bite)
It keeps the color of the cell more permanent
what are the steps of gram staining?
- 1. smear
- 2. air dry
- 3. heat fix
- 4. crystal violet (primary stain) 60 sec
- 5. water rinse
- 6. gram iodine (mordant)
- 7. water rinse
- 8. decolorizer (80% isopropyl alcohol + acetone)
- 9. water rinse
- 10. safranin (counter stain)
- 11. water rinse 1 min
- 12. blot dry
The acid-fast stain is a differential stain used to identify cells capable of retaining a primary stain when treated with acid alcohol.
What does acid fast positive contain?
Mycolic acid is a waxy substance which does not allow the cells to be stained by simple stains, but when stained by carbolfuchsin can retain this stain even acid alcohol decolorizer is used.
what are the three methods for acid fast staining?
- 1.Ziehl-Neelsen (ZN)
- 2.Kinyoun (K)
which uses heat to drive the carbolfuchsin in the cells
which uses a more concentrated, more lipid soluble form of carbolfuchsin.
There are three components in the acid-fast procedure. They are:
- 1. Carbolfuchsin
- 2. Acid alcohol
- 3. Methylene blue
A primary stain that is a phenolic compound that is lipid soluble.
Stains cells reddish purple.
A decolorizer that decolorizes non acid-fast cells.
A secondary stain that stains non acid-fast cells blue
PROCEDURE (Kinyoun Procedure)
1. Prepare a smear, air dry and heat fix.
2. Flood slide for 5-10 minutes with carbolfuchsin prepared with Tergitol No. 7 (heat is not required)
3. Decolorize with acid-alcohol (30 sec) and rinse with tap water. Repeat this step until no more color runs off slide.
4. Counterstain with alkaline methylene blue for 2 min. Rinse with water and blot dry.
5. Examine by brightfield microscopy using the 100X oil-immersion objective. Acid-fast organisms stain red; the background and other organisms (non-acid fast organisms) stain blue.
what is the purpose of Endospore staining?
A differential stain used to detect the presence and location of bacterial endospores.
Endospore-forming cells initiate differentiation into endospores in response to low nutrient conditions.An endospore is a dormant form of the bacterium that allows it to withstand harsh environmental conditions, even for hundreds or thousands of years.
is used to stain the spore because it has a low affinity for cellular material. Therefore,vegetative cells and endospore mother cells can be decolorized with water and counter stained with saffranin.
What is the purpose of the Starch Hydrolysis?
The purpose is to see if the microbe can use starch, a complex carbohydrate made from glucose, as a source of carbon and energy for growth.
What is the enzyme for starch?
What is the medium used in starch hydrolysis?
What is the reagent for starch hydrolysis?
iodine reagent is added to detect the presence of starch. Iodine reagent complexes with starch to form a blue color in the culture medium.
What was the result for starch hydrolysis?
- Bacillus subtilis (clear)
- Escherichia coli (Not clear)
If the area is clear starch is hydrolyzed
what is the purpose of casein hydrolysis test?
Casease is an exoenzyme that is produced by some bacteria in order to degrade casein. Casein is a large protein that is responsible for the white color of milk.
It is a source of carbon and energy and degrades into amino acids
What is the medium for Casein hydrolysis?
Milk a1% skim milk
what is the enzyme for casein hydrolysis?
what is the substrate for Casein hydrolysis?
Casein and protein
What is the reagent for Casein hydrolysis?
15% TCA Trichloroacetic acid
it reacts with starch and make it precipitate and react with protein and peptide to give white color precipitate
what is the results for casein hydrolysis test?
- Bacillus subtilis: clear
- Escherichia coli (not clear)
- if presence
- protein such as casein will be opaque
What is the purpose of Gelatin hydrolysis test?
The purpose is to see if the microbe can use the protein gelatin as a source of carbon and energy for growth
what is the enzyme for gelatin hydrolysis test?
What is the medium for Gelatin Hydrolysis test?
What is the substance for Gelatin hydrolysis test?
Gelatin and protein
what is the product of Gelatin Hydrolysis test?
what is the reagent for Gelatin hydrolysis?
sit for 15 minutes
What is the results for Gelatin Hydrolysis?
- Bacillus subtilis clear
- Escherichia coli not clear
what is the purpose of lipid hydrolysis?
Lipase allows the organisms that produce it to break down lipids into smaller fragments. Triglycerides are composed of glycerol and three fatty acids.
What is the enzyme for lipid hydrolysis?
What is the medium for Lipid hydrolysis?
pH indicator SPIRIT BLUE agar plate
How is the result for Lipid Hydrolysis?
- Escherichia coli clear (light blue)no lipid hydrolyzed
- Bacillus subtillis Dark blue (hydrolyzed)
What is the purpose of Hydrogen sulfide production?
This test determines whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism.
What is the medium for H2S?
SIM (Sulfide indole motility)
What is the enzyme for H2S?
What is the substrate for H2S?
What is the purpose of Catalase test?
The purpose is to see if the microbe has catalase, a protective enzyme capable of destroying the dangerous chemical hydrogen peroxide
what is the substrate for catalase test?
What is the reagent for Catalase test?
what is the product for Catalase test?
Water and Oxygen
what is the results for catalase test?
- Staphylococcus epidermidis (oxygen bubble)
- Enterocuccus faecalis (neg)
No bubble there is no catalse it means no oxygen making water
what is the purpose of Oxidase test?