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112 Cards in this Set
- Front
- Back
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Which bacteria is hard to stain?
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Spirilli
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For which staining is the slide not heat-fixed?
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The negative stain, using acidic dyes
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What composes the thick cell wall of Gram +ve bacteria?
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The thick cell wall is composed of peptidoglycan network associated with matrix substances, teichoic and teichuronic acids.
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What are the important factors in determining the Gram reaction of a bacterium?
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The physical mass and thickness of the cell wall, not the chemical composition of the cell wall.
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What does the decolourizing agent used in the Gram staining do?
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It dehydrates the cell wall, causing the cell wall to collapse, and prevents the extraction of the initial crystal violet dye in the Gram-positive bacterium.
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Gram staining is what kind of a phenomenon?
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It is a physical phenomenon.
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In what cases would Gram-staining not give clear-cut results?
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If Gram-positive bacterial cultures were too old, they would stain Gram-negative.
Also some species of bacteria are inherently Gram-variable. |
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What kind of cells must be used in Gram staining?
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Young, vigorous cultures.
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What must be done when dealing with Gram-variable cells?
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Prepare the Gram stain side by side on the same slide with a known culture.
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How is a Gram stain prepared? Using what agents?
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1. Cells are heat fixed on slide.
2. Primary stain - Modified Hucker's crystal violet, 1min. 3. Wash with tap water 4. Mordant - Gram's iodine solution, 1min. 5. Wash with tap water. 6. Decolourizing agent - 95% ethyl alcohol/absolute alcohol. 7. Wash with tap water 8. Counterstain - Safranin, 1min |
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What colour would a Gram +ve cell exhibit? A Gram -ve cell?
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Gram +ve: purple
Gram -ve: red |
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What does a simple stain let us observe?
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A cell's shape, size, and arragement
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What are the two kinds of dyes?
Which one is more commonly used, and what is it composed of? |
Natural and Synthetic.
Synthetic is more commonly used; aniline/coal-tar dyes, which are derivatives of benzene. |
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What is a basic dye composed of? An acidic dye?
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Basic dye: +ve choromophore
Acidic dye: -ve chromophore |
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What is a basic dye?
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chloride or sulfate salt of a coloured base that makes a +ve chromogen
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What is an acidic dye?
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Na, K, Ca, or ammonium salt of a coloured acid that makes a -ve choromogen
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what kind of dyes are crystal violet, carbol fuchsin, and methylene blue?
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Basic
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What dye is used in a negative stain?
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Nigrosin
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Which genus of bacteria requires an acid-fast stain? Why?
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Mycobacterium, as they do not stain readily by basic staining procedures ex. Gram-stain.
This is because these bacteria have waxy cell walls with large amounts of lipoidal material. These walls are hydrophobic, and are thus impermeable to stains and other chemicals in aqueous solution. |
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What are the three agents used in acid-fast staining?
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1. Strong, red, basic dye: basic fuchsin. Dissolved in an aqueous solution containing phenol, this is called Ziehl' carbol fuchsin
-Heat treated 2. Decolourizing acid-alcohol 3. Methylene blue |
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Procedures of acid-fast staining?
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1. Flood with Ziehl's carbol fuchsion, steam over flame for 3-5 minutes.
2. Wash with tap water, decolourize with acid-alcohol for 10-30 seconds. 3. Apply methylene blue for 30-45 seconds, wash, drain, and blot dry. |
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What extra procedure is required in acid-fast staining, in preparing the initial slide of the sample culture? Why?
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The smears of the bacteria must be prepared in a small drop of serum of egg albumin.
The protein ehances the adherence of the bacteria to the glass surface, and also provides a material for light background staining. |
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What colour is an acid-fast bacteria? Non-acid-fast?
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Acid-fast: Red
Non-acid-fast: Blue |
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The better the ability to bend light, the better the...
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The magnification
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What does the condenser to on a microscope?
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It focuses the light on the specimen
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What does the iris diaphragm do?
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it controls the amount of light passing through the condenser
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What kind of microscope remains in focus when the objective lens are changed?
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A parfocal microscope
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What is resolution? What is the resolution of the human eye? Of the bacteria? Of the microscope?
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It is the ability to show closely adjacent objects as distinct.
Human eye: ~0.1mm Bacteria: 0.001mm = 1 micron light microscope: 0.2micrometers |
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Magnification vs. Resolution?
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You know.
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What are the two kinds of microscopes? Their subgroups?
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Light microscopes
1. bright field 2. dark field 3. phase contrast 4. fluorescent Electron microscope 1. transmission EM 2. scanning EM |
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Equation of resolution?
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resolution = 0.5 lambda / Numerical Aperture
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What is the numerical aperture proportional to?
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The refractive index of the medium the light passes through
Refractive index is the ability to bend and collect light |
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why is immersion oil used?
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it has a higher refractive index, thus allows higher resolution.
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What are the two kinds of fixation and what do they allow?
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Heat fixation preserves the overall morphology of the cell, but not the fine internal structures.
Chemical fixation preserves both fine cellular structures and the overall morphology |
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The three things a staining does
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1. preserves the specimen
2. increases visibility of the specimen 3. accentuates specific morphological structures. |
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How thick is the peptidoglycan layer of the Gram +ve cell? of a Gram -ve cell?
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Gram +ve: 20-80nm
Gram -ve: 2-7nm |
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When and why are spores formed?
What bacteria form spores? |
They are formed in response to unfavourable environmental conditions, in efforts to protect the bacterial DNA.
Species in the genera Bacillus and Clostridium form spores. Only some Gram +ve cells form spores. |
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where does the spore form? What is its technical term in account for this?
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The spore develops within the mother vegetative cell, and is call an endospore.
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What is the stain used for spore staining?
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Malachite green
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What are the different positions of an endospore within its mother vegetative cell?
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(Free spore)
Terminal Subterminal Central Swollen sporangium |
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What is the spore staining procedure?
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1. Heat fix
2. Malachite green, steam for 2-3 min. 3. Wash with tap water 4. Safranin solution for 30s 5. Wash with tap water, drain, blot dry. |
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what is a capsular material composed of?
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Polysaccharides
Glycoprotein Polypeptide |
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What is the capsular membrane called when it is discrete? When it is amorphous?
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discrete: capsule
amorphous: slime layer |
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What affects the size of the capsule?
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The species/strain of the bacteria, and the medium on which it was cultivated
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What is the function of the capsule?
What does its presence indicate? |
Resistance to dessication
Adhesion to favourable sites The capsule protects the cell from being phagocytized by the host white blood cells. Its presence usually means the bacteria is in its virulent form, capable of causing disease. |
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What is the Quellung phenomenon?
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It is the swelling of the capsule in reaction with specific antibodies.
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What is it common that the bacteria and its surrounding is stained more intensely than the capsule?
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The capsular materials are water-soluble and uncharged; thus simple staining will not adhere to them.
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What is the capsular swelling called?
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Quellung phenomenon
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How will a capsule staining appear?
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The bacteria stained blue by methylene blue
The background stained black by nigrosin The capsule colourless |
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what agent is used in capsule staining?
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the nigrosin stain
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What is the procedure in preparing a capsule stain?
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Same as negative stain
Then fix the smear with absolute alcohol for 2 min. Drain and let dry. Stain with crystal violet for 2 min. Wash with tap water, drain, air dry VERTICALLY before examining with oil immersion |
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What shape is a flagella? How does it move?
Relative size to the bacteria? |
It is coiled into rigid spirals that revolve around their points of attachment.
Much longer than the bacteria, but also much thinner in diameter, 0.01 - 0.05 micrometers |
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The flagella is 10 times smaller than the limit of resolution of the light microscope. How can they be observed?
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The thickness of the flagella can be increased by coating them with MORDANTs, ex. tannic acid and potassium alum.
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How are motile bacteria observed? Directly vs. Indirectly?
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Directly, in wet preparations, where the bacteria are able to move freely.
Indirectly, by inoculating them into a motility-agar medium, in which they are able to migrate/disperse away from the line of inoculation |
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How is a wet preparation done?
What is this called? |
Gray's method
1. Incubate bacteria in water, allow flagella to extend fully 2. Make a film and air dry 3. Flood with mordant for 10min. 4. Rinse GENTLY 5. Flood with Ziehl's carbol fuchsin for 5 min, rinse gently 6. Air dry |
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How is a hang-drop motility test done?
What is crucial in this approach? Where to focus? |
1. Use petroleum jelly and a cover-slide to invert a drop of the bacteria in saline solution over a slide.
2. Observe using the high-power objective (40X) Proper illumination is proper, as this is an unstained preparation. Focus on the edge of the drop, where the organisms are more active due to greater oxygen supply. |
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What is different about the motility-agar medium?
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It has a lower concentration of agar than normal agar medium, at 0.35%
This semi-solid state allows motile bacteria to move away from the line of innoculation |
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Different positions of the flagella?
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Atrichous
Monotrichous Amphitrichous Lophotrichous Peritrichous |
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What is Brownian motion?
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Random movement of microscopic particles in a fluid
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What is the mechanism of the cell-wall stain?
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Cetylpyridinium chloride dissociates in water to make:
+ve charged cetylpyridinium cation -ve charged chloride anion the cation attaches to and neutralizes the negatively charged cell wall. Cell wall then becomes +vely charged as more cations add to it The cell wall is not +vely charged, and an acidic dye (with a negative chromophore) can be used to stain it. |
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What are the dyes/agents used in the cell wall stain?
Procedures? |
1. Heat fix
2. 3 drops of cetylpyridinium chloride 3. 1 drop of Congo red 4. Rotate the drops with rotating motion while holding the slide in hands, for about 60 seconds 5. Rinse with tap water 6. Counterstain for about 60 seconds with Ziehl-Neelson methylene blue 7. Rinse and blot-dry |
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What is a defined medium?
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Medium for which exact composition is known
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What is a minimal medium?
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Medium that has just enough ingredients to support growth, usually a carbon source and salts
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What is an enriched medium? What is it used to grow?
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Used to grow fastidious heterotrophs, such as pathogens
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What is a selective medium?
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favours the growth of certain organisms
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What kind of medium is a MacConkey agar?
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It is a selective medium for Gram -ve bacteria
It is also a differential medium to differentiate bacteria on basis on lactose fermentation |
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What kind of medium is a deoxycholate medium?
What are its components? How does it work? |
It is a selective medium for Gram -ve bacteria
Sodium deoxycholate inhibits Gram +ve cells, by destroying integrity of cell membrane. It is a surface-active detergent, and acts by disrupting the interaction between membrane proteins and lipids. Sodium citrate is also present, required to potentiate activity of deoxycholate It is also a differential medium to differentiate bacteria on basis of lactose fermentation |
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what indicator is used for deoxycholate mediums?
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neutral red
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What kind of medium is phenylethyl alcohol medium?
How does it work? |
It is a selective medium for Gram +ve bacteria
phenylethanol inhibits Gram -ve, by interfering with DNA synthesis and cell wall integrity |
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What is a differential medium?
what are some examples? |
Medium that distinguishes between different groups of microorganisms, based on their biological characteristics
Blood agar: hemolytic versus non-hemolytic MacConkey agar: lactose fermenters vs nonfermenters |
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What are the two kinds of ways a cell can be lysed?
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membrane disrupting exotoxins:
pore-forming exotoxins phospholipases |
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What is the pH range/colour of neutral red indicator?
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Red below pH 6.8
Yellow above pH 8.0 |
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Are selective media 100% selective?
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No!
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What are the toxic oxidation products?
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Superoxide radical
Hydrogen peroxide Hydroxyl radical |
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What enzymes can detoxify the toxic oxidation products?
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Superoxide dimultase
Catalase Peroxidase |
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What are the three criteria for anaerobic bacteria?
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1. Nonformation of oxidation products during preparation
2. Exclusion form oxygen during incubation and cultivation 3. Low O/R potential |
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What is the O/R potential?
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oxidation productions
------------------- reduction products |
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Why should a culture in liquid media never be used as the sole method for isolating anaerobes from samples?
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Recovery of anaerobes in liquid media is generally poorer.
Quantitation is not possible. |
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What are the two methods of anaerobic cultivation?
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Anaerobic chamber
Anaerobic Jar |
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What are the crucial components of an anaerobic jar?
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Catalyst: palladium
GasPak envelope: Sodium bicarbonate pallet x1 Sodium borohydride pallet x1 Powdered citric acid |
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What does the GasPak envelope generate? What for? How?
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It generates H2 and CO2
The H2 reacts with O2 in the jar to form H2O, thus removing O2 from the atmosphere CO2 supports the growth of organisms requiring it Addition of water to the envelope reacts with the oxygen in the presence of the catalyst to provide an anaerobic atmosphere |
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What does the polysaccharidase do?
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It hydrolyzes polysaccharides to saccharides and sugars
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What does the lipdase do?
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It hydrolyzes lipids to glycerol and fatty acids
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What does the protease do?
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It hydrolyzes proteins to peptides and amino acids
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How is starch hydrolysis tested?
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Gram's iodine solution is added. If starch is present, the two react to form a dark blue colour.
No colour formation indicates hydrolysis of starch |
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What is the lipid used in the lab, for lipid hydrolysis test?
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Tributyrin, a triglycerol
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What is the protein used in the lab, for protein hydrolysis test?
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Casein, the principal nitrogenous constituent of milk.
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How are anaerobes grown in liquid culture?
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By the addition of reducing agents:
Sodium thioglycolate Cysteine (amino acid present in cooked meat medium) |
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Why is a cooked meat medium used?
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Cooked meat contains amino acid cystein, which is a reducing agent
The solution is thus anaerobic |
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What are the reducing agents added to liquid medium?
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Cysteine
Sodium thioglycolate |
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What kind of enzymes are those that can break down large complex molecules?
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Exoenzymes, extracellular
Sent out to break down complexes to make them easier to transport into the bacteria |
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What is the principal compound that stores energy in cells?
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ATP
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What is the overall scheme of catabolism?
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electron donor becomes oxidized, and transfers electron to an electron acceptor which gets reduced.
this process provides the energy to generate ATP from ADP |
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What occurs in aerobic respiration?
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electron donor becomes oxidized, and transfers electron to O2, an electron acceptor, which gets reduced to H2O
this process occurs through ETC, Electron Transport Chain, and provides the energy to generate ATP from ADP |
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What occurs in anaerobic respiration?
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Same process as aerobic, but the electron acceptor is not O2, but NO3-, SO4-, etc
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What is crucial to be supplied in respiration?
How are they supplied? |
The final/terminal electron acceptor must be present to carry out respiration.
It may be oxygen under aerobic conditions, or the media addition of nitrate, sulfate, etc for anaerobic respiration |
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What occurs in fermentation?
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An organic compound already present in the bacteria is reduced,
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What mechanism does fermentation use?
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Substrate level phosphorylation
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What are the end products of fermentation?
How are they detected? |
CO2 alone or mixture of H2 and CO2.
H2 is insoluble and could be detected using a Durham tube |
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What indicator is used for carbohydrate fermentation?
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Phenol Red pH indicator:
Yellow below pH 6.6 Red above pH 8.0 |
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How is a carbohydrate fermentation tube prepared?
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- nutrient broth with 0.5% of the particular carbohydrate
- Durham tube - pH indicator |
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What is the Hugh and Leifson test for?
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It is to test oxidative vs. fermentative metabolism
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How is a Hugh and Leifson test prepared?
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- broth culture
- layer of sterile melted petrolatum/sterile paraffin oil - pH indicator |
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What pH indicator is used in the Hugh and Leifson test?
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Bromothymol blue pH indicator:
Yellow below pH 6.0 Green at pH 7.0 Blue above pH 7.6 |
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Why is a bromothymol blue pH indicator useful?
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It detects even a slight change in pH
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How is a Hugh and Leifson test 'tweaked'?
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The sugar (glucose) concentration is increased to 1%
The amount of peptone (which can neutralize some acid) is decreased |
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What carbohydrate is used in the Hugh and Leifson test?
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Glucose
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What unique characterization allows detection of hydrogen sulfide gas?
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its smell of rotten eggs
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How is the formation of hydrogen sulfide detected?
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By the formation of black metal sulfide precipitate, which forms when H2S reacts with heavy metal salts
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How is H2S produced from cysteine?
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cysteine + water -> pyruvic acid + hydrogen sulfide + ammonia
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What does a culture media used for detection of hydrogen sulfide production contain?
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peptone and thiosulfate, sources of sulfur
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What is used at the indicator in hydrogen sulfide production?
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Iron (ferric and ferrous salts), which react with H2S to form iron sulfide, a black precipitate.
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