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What are the steps to heat fix bacteria to a slide from an agar medium?
- 1. Add a drop of water to a slide.
- 2. Remove small amount of bacteria from culture with loop, and touch the drop of water 2-3 times.
- 3. Flame the loop.
- 4. Using loop spread suspension over entire slid.
- 5. Let air dry completely.
- 6. Pass slide through flame 6 times, let cool, and repeat.
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What are the steps to heat fix bacteria to a slide from broth medium?
- 1. Aseptically place 2-3 drops of the culture to clean slide
- 2. Using loop spread suspension over entire slid.
- 3. Let air dry completely.
- 4. Pass slide through flame 6 times, let cool, and repeat.
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Are bacteria positively or negatively charged?
Negative
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If the color portion of the dye resides in the positive ion it is referred to as a/an ______?
Basic Dye
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If the color portion of the dye resides in the negative ion it is referred to as a/an ______?
Acidic Dye
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The absorption of dye by the bacterial cells is called a/an ________ stain.
Direct
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Do basic or acidic dyes direct stain bacteria?
Basic
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The non-absorption of dye of a bacterial cell, but dye is deposited around the organism is called a/an _______ stain?
Indirect
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Do basic or acidic dyes indirect stain bacteria?
Acidic
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Safranin stain is a basic or acidic stain?
Basic
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Methylene Blue is a basic or acidic stain?
Basic
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Nigrosin is a basic or acidic stain?
Acidic
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What are the steps to direct stain bacteria on a slide?
- 1. After heat fixing bacteria to slide cover entire slide with a basic stain.
- 2. Leave for 1 minute.
- 3. Using water bottle wash off excess stain.
- 4. Blot dry with blotter paper.
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What are the steps to indirect stain bacteria on a slide?
- 1. Add a drop of acidic stain to a slide.
- 2. Aseptically mix bacteria to stain.
- 3. Using another slide spread mixture across slide.
- 4. Let dry. (Do not heat fix.)
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Why is stain not washed off on and indirect bacterial stain?
Because you are staining the background and not the bacteria. If stain is removed from background you will not be able to see bacteria.
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Why is gram stain called a differential stain?
Because it can differentiate between Gram + and Gram - bacteria.
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What are the characteristics of a Gram + bacteria cell wall?
Single thick layer of peptidoglycan. 60-90% of Gram + cell wall is peptidoglycan.
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What are the characteristics of a Gram - bacteria cell wall?
Very thin later of peptidoglycan surrounded by a layer of phopholipids, lipoproteins, and lipopolysaccharides. Only 10-20% of Gram - cell wall is peptidoglycan.
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What are the steps of a gram staining procedure?
- 1. Bacteria are first stained with the basic dye crytal violet. Both G+ and G- become directly stained.
- 2. Bacteria are then treated with Gram's Iodine solution. Allows stain to be retained better by forming insoluable crystal blue-iodine complex.
- 3. Gram's Decolorizer, a mixture of alcohol and acetone, is then added. This is the differentiating step.
- 4. Finally, the counter stain safranin (a basic dye) is applied. G+ are already stained and do not absorb pink safranin.
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Why do Gram + bacteria retain the crystal violet-iodine complex?
In the Gram + bacteria, the crystal violet and iodine combine to form a larger molecule that precipitates out within cell. The alcohol/acetone mixture then causes dehydration of the multilayered peptidoglycan, decreasing space between molecules and trapping the crystal violet-iodine complex.
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Why do the Gram - bacteria become decolorized?
The alcohol/acetone mixture is a lipid solvent, that dissolves the outer cell wall layers. The very thin layer of peptidoglycan that is left is unable to retain the crystal violet-iodine complex.
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What are 3 conditions that may cause G+ bacteria to stain as G-.
- 1. The method and technique used. (overheating during heat fixation, over decolorizing w/ alcohol, and over washing)
- 2. Age of the culture.
- 3. The organism itself. Some G+ bacteria are more able to retain the crystal violet-iodine complex than others.
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Some bacteria produce capsules. What is the chemical composition of capsules?
Polysaccharide, polypeptide, or both.
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What is the function of a bacterial capsule?
- Resist phagocytosis.
- Store energy reserves.
- Prevent being engulfed by protozoans.
- Adhesion to surfaces, prevents being flushed out.
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What are the 2 genera of bacteria that produce endospores?
Bacillus and Clostridium
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What is the function of endospores?
Survival in harsh environments. Endospores are virtually indestructable.
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What is the characteristic appearance of a Clostridium bacteria that contains an endospore?
It has a "tennis raquet" appearance.
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What are the 5 flagellar arrangements?
- Monotrichous
- Amphitrichous
- Lophotrichous
- Peritrichous
- Axial filaments (spirochetes)
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Define monotrichous.
A single flagellum at one pole.
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Define amphitrichous.
A single flagellum at both poles.
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Define lophotrichous.
2 or more flagellum at one or both poles of the cell.
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Define peritrichous.
completely surrounded by flagella.
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What is the chemical composition of flagella?
Flagellum is a noncontractile, semi-rigid, helical tube composed of protein (Flagellin).
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What is the function of flagella?
Motility
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What is taxis?
Taxis is a motile response to an environmental stimulus and functions to keep bacterial in an optimum environment.
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What are 3 methods to detect bacterial motility?
- Direct observation using special-purpose microscopes.
- Motility test medium.
- Flagella staining.
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What are 2 types of microscopy used in direct observation of motile bacteria?
- Phase-contrast microscopy - gives the effect of a direct staining, flagella are darker than the background.
- Dark-field microscopy - gives effect of an indirect stain, flagellum apprears bright against a dark background.
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Describe Motility Test medium.
Semi-solid with low agar concentrations as to not hinger motility. Used as a stab tube.
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Describe the results of a non-motile organism that has been cultured in a stab tube with motility test medium.
Growth occurs only along the line of inoculation.
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Describe the results of a motile organism that has been cultured in a stab tube with motility test medium.
Growth occurs throughout the tube. Growth along the stab line appears cloudy as it moves away from stab line.
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What are bacterial enzymes made of?
Proteins
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How do enzymes differentiate between bacteria?
Different species of bacterium have different DNA. DNA codes for protein synthesis, thus different DNA codes for different enzymes. Enzymes catalyze various chemical reactions that the organism is capable. This means different bacteria carry out different and unique sets of biochemical reactions.
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What is an Exoenzyme?
An enzyme produced be a bacteria and secreted into surrounding area to breakdown larger nutrients so they can enter the bacteria.
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What is an Endoenzyme?
Enzymes used within the bacteria cell.
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What is the name of the exoenzyme used to breakdown starch called starch hydrolysis?
Diastase
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What is the appearance of a diastase producing bacteria that has been inoculted, incubated, and flooded with iodine on a starch agar plate?
The iodine will stain starch a dark brown color. The area around the bacteria will be devoid of starch and will not stain, appearing clear and see through on the plate.
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What is the name of the exoenzyme that breaks down the milk protein casein called protein hydrolysis?
Protease
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What is the appearance of a protease producing bacteria that has been inoculted and incubated on a skim milk agar plate?
If casein is hydrolyzed, there will be a clear zone around the bacterial growth. If casein is not hydrolyzed, the agar will remain white and opaque.
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In the lab E. coli and B. subtilis were cultured on starch and skim milk agar plates. Which one of these bacteria produced diastase and which one produced protease?
B. subtilis produced both diastase and protease. E. coli produced neither enzyme.
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