Lab 3 Flashcards
What is the standard plate count procedure?
- Used to enumerate bacteria
- A specified volume is plated and incubated
- Resulting colonies counted
What are two required properties a diluent for a bacterial dilution?
- Must have osmolarity like that of sample
- Should not support bacterial growth
What are the two important potential sources of error in preparing dilutions?
- Sampling error resulting from an unequal distribution of bacterial in original sample or dilution blanks (always mix fluids well prior to sampling to avoid this)
- Inaccuracy in pipetting; more transfers = greater chance of error (use as few dilutions as possible to avoid this)
Describe King B agar.
- Typical for Pseudomonas, which produce fluorescent pigments.
- Recommended for production and detection of fluorescent pigments
- Magnesium chloride and potassium sulfate in the media promote production of pyocyanin, a blue or blue-green, fluorescent pigment.
- Glycerol provides a carbon source and assists in pyocyanin production.
- The pigments diffuse from colonies into the agar and can be observed under UV light.
What is the result of an oxidase test if the bacteria is Pseudomonas?
Usually positive
Describe nitrate reduction broth.
- Many bacteria reduce nitrate salts to nitrite or nitrogen gas under anaerobic conditions.
- The nitrate molecule acts as an electron acceptor.
- Following incubation, nitrite accumulation can be detected by addition of : alpha-napthylamine and sulfanilic acid which react with nitrite to produce a compound that appears red
- Some species, like Pseudomonas further reduce nitrite to ammonium; this gives a false negative
- A negative test is confirmed by adding a pinch of zinc; if unreduced nitrate is present, it will produce a red colour (i.e., the zinc reduces nitrate to nitrite which will then react with the previous reagents)
- If after adding zinc, there is no colour change, this would be considered a positive nitrate reduction test
Describe Bile Esculin Azide Agar.
- Contains bile to inhibit growth of some bacteria (e.g., non-enteric).
- Useful for identification of Enterococci
- Bacteria that can hydrolyze esculin release glucose and esculetin.
- Esculin reacts with iron salts (ferric citrate) in the medium and form dark brown to black complexes
How does the Gram stain procedure differentiate bacteria?
- Gram-negative have an outer membrane and a thin peptidoglycan layer which loses the primary dye when flooded with alcohol
- Gram-positive have a thick peptidoglycan layer that retains the primary purple dye and no outer membrane
Describe what over or under decolorization can result in during a gram stain.
- Under decolorization can result in a false gram positive result
- Over decolorization can result in a false gram negative result
Describe how nutrition affects bacterial growth.
- Bacteria also need macronutrients (C H O N P S) and micronutrients (K, Ca, Mg, Fe), trace elements (Cl, Na, Zn, Mn, Mo, Ni, Cu…).
- The nutrients need to be in a form that bacteria can use (growth depends on nutrient availability).
- For example, the air is 78% nitrogen (as N2), but very few bacteria can use N2 as a source of nitrogen for making amino acids, nucleotides.
Describe the growth phases of bacteria.
Lag: adaptation to new media and/or growth conditions, ‘gearing’ up to take advantage of nutrients
Exponential: cells are growing, dividing, cell numbers (concentration) are increasing
Stationary: cells are adapting to poor environmental conditions, low nutrients, high waste
Death: cells are dying
Describe how temperature affects bacterial growth.
- Growth depends on the preferred temperature of the bacteria of interest.
Describe how pH affects bacterial growth.
- pH represents the concentration of H+ ions which contributes to the availability of other nutrients such as iron
Describe how oxygen affects bacterial growth.
- Some bacteria:
- have absolute requirement for oxygen
- cannot tolerate oxygen
- are somewhat indifferent to oxygen
Obligate aerobes.
Need oxygen
Obligate anaerobes
Oxygen is toxic
Facultative anaerobes
Prefer oxygen, but can grow in low oxygen conditions.
Aerotolerant anaerobes
Prefer no oxygen, but can grow in its presence.
Microaerophiles
Require small amounts of oxygen
Need oxygen
Obligate aerobes.
Oxygen is toxic
Obligate anaerobes
Prefer oxygen, but can grow in low oxygen conditions.
Facultative anaerobes
Prefer no oxygen, but can grow in its presence.
Aerotolerant anaerobes
Require small amounts of oxygen
Microaerophiles
What are colony forming units?
CFU = the concentration (# of cells in a given volume) can be approximated using a colony count method.
CFU = a unit that estimates the # of cells in a sample that are (1) viable, (2) able to multiply via binary fission.
Why does CFU only provide approximate counts?
- 1 colony may not have arisen from 1 cell
- We’re only counting living cells that can grow in the environment provided
- CFU assumes that every colony is separate and founded by a single viable microbial cell, which may not be the case.
What can the gram stain identify? [4]
- Shape of bacteria
- Arrangement of bacteria
- Nature of the cell wall
- Presence of spores
Compare a gram stain of a pure versus mixed culture.
Describe the gram-stain process.
- Flood the slide with Crystal violet. Wash off with water after 1 minute.
- Flood the slide with iodine-mordant – holds CV-I complex in cell membrane. Wash off after 1 minute.
- Decolorize slide with ethanol.
- Counter-stain with safranin or aqueous fuschin. Wash off with water after 1 minute. Tap of excess water and let slide dry.
Describe the gram-stain process.
- Flood the slide with Crystal violet. Wash off with water after 1 minute.
- Flood the slide with iodine-mordant – holds CV-I complex in cell membrane. Wash off after 1 minute.
- Decolorize slide with ethanol.
- Counter-stain with safranin or aqueous fuschin. Wash off with water after 1 minute. Tap off excess water and let slide dry.
Describe the first step of the gram stain process.
- Flood the slide with Crystal violet.
- Crystal violet is positively charged and binds to the negatively charged cell wall structures. In this step, all cells are stained purple
Describe the second step of the gram stain process.
- Flood the slide with iodine-mordant. Iodine (I) molecules is a “dye-fixator” or mordant.
- Iodine molecules binds to CV molecules (via non-covalent bonds) and form large insoluble complexes (CV-I) in the peptidoglycan and inner membrane.
- In this step, all cells are still stained purple.
Describe the third step of the gram stain process.
- Decolorize slide with ethanol. Alcohol dissolves the outer membrane of Gram-negative and disrupts the thin peptidoglycan layer so that Crystal-violet-iodine complexes escape.
- In Gram positive cells, Crystal-violet-iodine complexes are retained.
- Gram-positive bacteria are still purple, Gram-negative bacteria are colourless.
Describe the last step of the gram stain process.
- Counter-stain with safranin or aqueous fuschin.
Safranin is used to counter stain the Gram-negative bacteria that were colourless in step 3, can now be visualized as pink. - Safranin is a positively charged stain that binds to the negatively charged cell wall structures in Gram negative bacteria.
When gram-staining an unknown bacteria, is it important to have a positive control (a sample of bacteria that is known to be Gram-positive) and a negative control (a sample of bacteria that is known to be Gram-negative).
Why?
- To ensure the gram-stain procedure was conducted correctly
What could possibly go wrong in a gram stain procedure?
- Contaminated sample
- Overheating; not heating enough heating
- Stain for too long
- Overwashing/destaining
- Too heavy a smear
- Too old a culture
- Not washing long enough with ethanol
- Washing too long with ethanol
- Using reagents in the wrong order
Describe a compound light microscope.
- Uses visible light
- A condenser lens focuses the light onto the specimen
- The objective lens picks up the light transmitted by the specimen and magnifies the image
- The image is viewed and further magnified through eye pieces.
Example application: viewing cells after gram-staining; looking for somatic cells in a raw milk sample
Describe the magnification of compound light microscopes.
- 10X - difficult to view bacteria
- 40X - better, but bacteria are small
- 100X - best viewed - requires immersion oil
How is actual magnification calculated?
objective x eyepiece
What is resolution?
- Resolution is the ability of the microscope to distinguish between 2 objects/detail. Shortest distance between 2 points on a specimen that can be distinguished.
- At higher magnifications, the ability to “resolve” 2 points is dependent on the light and the objective lens (numerical aperture).
Why do oil immersion objective lenses improve resolving power?
- Oil has a high refractive index - it will ‘bend’ light back toward the lens
What plates are considered valid for counting (via CFU) in this class?
Those that have between 30 - 300 colonies
