Laboratory Identification of Microorganisms

Question	Answer
In creating a slide, why is it necessary to dry cells in air before heat fixing?	So that the cell shape is not altered or disrupted as proteins are oxidised rather than coagulated.
Why do many colonies appear pigmented?	Microorganisms may produce pigmented substrates or waste products. If these are water soluble they may spread out through the culture medium. The production of pigments is highly variable, and may be dependent on temperature or the availability of a certain nutrient.
What is the importance of Gram-staining unknown specimens?	It will only stain bacteria purple or red (some yeasts develop a golden 'halo' but these are easily distinguishable) and so it visualises bacteria but also differentiates between mixed populations of bacteria.
Are there any bacteria that do not Gram-stain, and if so why not?	A rare few species don't because they do not possess a cell wall and so there is no peptidoglycan present to bind to a dye.
Why are endospores difficult to stain?	They possess many protective layers, including a plasma membrane, a germ cell wall, a peptidoglycan cortex, an inner spore coat and an exosporium, which are designed to protect the spore and its genetic material from damage and so are impervious to the different stains of the Gram stain.
How do Gram-positive and Gram-negative bacteria protect themselves with their cell walls?	The thick peptidoglycan layer in Gram-positive bacteria is anchored to the plasma membrane with lipoteichoic acid and anchored together by teichoic acid which confers strength and physical protection. The envelope of Gram-negative bacteria is impermeable and so provides protection by preventing infection or diffusion of toxins/antimicrobial agents.
What is catalase and what is its function?	An enzyme which detoxifies H2O2 to gaseous O2 to protect against cellular damage by the free radical that is produced in aerobic respiration. Catalase action is a defensive mechanism.
What are the selective and differential agents in mannitol salt agar?	7.5% NaCl and mannitol (sugar) and phenol red, respectively.
What are the selective and differential agents in Horse Blood Crystal Violet agar?	Crystal violet and fresh blood, respectively.
Which microorganisms does mannitol salt agar select for?	Mannitol-fermenting saprophytic pathogenic staphylococci.
What goes on in a catalase biochemical test?	A sterile inoculating loop of a culture growth is inserted into a glass tube of a few drops of H2O2. If there is effervescence, O2 is being produced and catalase is present.
Why should a catalase test not be performed on bacteria growing in a fresh blood agar?	Because blood contains catalase and may give a false positive result.
What are the selective and differential agents in MacConkey agar?	Bile salts, and lactose and neutral red, respectively.
Which organisms do bile salts select for?	Enteric microorganisms.
What are the selective and differential agents in Rose Bengal Chloramphenicol agar?	Chloramphenicol and Rose Bengal, respectively.
What does chloramphenicol select for?	Yeasts and moulds, but it inhibits everything else.
Name a common catalase positive and catalase negative bacterium.	Staphylococcus species are generally catalase positive, whereas Streptococcus species are generally catalase negative.
What is the oxidase test?	The oxidase test is a biochemical assay to determine whether cytochrome oxidase, the last enzyme in the bacterial and mitochondrial electron transport chains, is present. If it is present then the organism is an aerobe and if it is not present then the organism is an anaerobe.
How does one perform an oxidase test?	The tip of a small spatula is covered with crystals of oxidase reagent and 10mL of sterile distilled water is added to it. This is pipetted on to a piece of filter paper and some colonies are transferred on to it using a Pasteur pipette. A blue colour will be produced within 10 seconds if an organism possesses cytochrome oxidase.
Why must the reagent for the oxidase test be made fresh just before the test?	The reagent is strong and rapidly oxidises in the air and turns purple, so it would produce a false positive result.
What is the oxidation-fermentation test?	A culture of bacteria is pushed to the bottom of a bijou bottle filled with glucose solution to determine their glucose usage. If they are anaerobes, a colour change (darker) will appear only at the bottom. If they are aerobes, a colour change will appear only at the top. If they are facultative anaerobes, a colour change will appear throughout.
As well as differentiating oxidative/fermentative glucose use, what else can the oxidation-fermentation test be used to determine?	The motility of an organism, as motile organisms will require more glucose.
Why is it important to perform a multitude of morphological and biochemical tests on an unknown sample?	Because species are unlikely to be distinguished on the result of one test alone, given that the tests are for widespread characteristics, so a profile can be built up which may then be definitive.
How does one perform a spore stain?	Place a slide with a heat fixed film over a boiling water-bath and cover the film with a small square of filter paper. Flood this with Malachite Green stain and steam for 10 minutes, adding more stain to stop the sample from drying out. Remove the filter paper and wash the slide with tap water before flooding with safranin (to counter stain for vegetative bacteria) and wash with tap water.
What colour do spores appear when stained?	Green.
What colour do Pseudomonas aeruginosa colonies appear?	Green.
What are the steps of performing a Gram stain?	Place the sections in xylol then down to water; wash slides with crystal violet which binds to peptidoglycan and then rinse with tap water; place in Lugol's iodine to fix retained crystal violet in Gram-positive bacteria and wash with water; differentiate in acetone alcohol until no more colour leaves the section; counter stain with safranin before rinsing in tap water. Each step should take around 20 seconds, then rinsing.
How do Gram-positive bacteria appear when Gram-stained?	They appear strongly purple, even after washing with acetone alcohol, as there is so much peptidoglycan for crystal violet to bind to.
How do Gram-negative bacteria appear after being Gram-stained?	The acetone alcohol washes the crystal violet out of the 2nm peptidoglycan layer in Gram-negative bacteria so that safranin stains them pink/red afterwards.

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Laboratory Identification of Microorganisms

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	Created by sophietevans over 11 years ago