Micro Lab Midterm

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when subculturing it is necessary to flame the loop before and after each inoculation to

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maintain the sterility of the instrument between inoculations

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When subculturing hold caps in your hand to

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prevent contamination via lab surfaces

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When subculturing the instrument before obtaining inoculum so as not to

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fix samples or kill organisms

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When subculturing, flame the neck of the tube before and after inoculating to prevent

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transfer of microbes from the neck/air to the nutrient agar

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the purpose of subculturing is

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to separate microbes for testing or to prepare and maintain stock cultures

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the sources of microorganisms found in the lab are

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those microbes naturally present in the environment or brought there by hosts

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a bacterial colony is

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a visible mass of microorganisms resulting from the mother cell, a clump of bacteria that are genetically alike

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does a bacterial colony increase in diameter when incubated

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yes

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you should be concerned by airborne pathogens because

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they are ubiquitous, can be pathogenic, and can contaminate lab work

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to control "lab contaminants"

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care should be taken in aseptic technique and handling of dishes/slides/slants/etc

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Iris diaphragm

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used to regulate the amount of light entering the lens system

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ocular lens

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found in the eyepiece and is part of the 2-lenssystem

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mechanical stage

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allows for movement of the slide using controlknobs

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nose piece

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contains the objective lenses and allows fortheir rotation

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condenser

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collects and concentrates light as it passesupward from the light source

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streak technique

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simple staining

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Shows morphology and arrangement of cells. Uses basic dyes since they are positivelycharged and are attracted to the slightly negatively charged bacteria.Methylene blue, Crystal Violet, and Carbolfuchsin

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negative staining

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Uses an acidic stain (Nigrosin or India ink). Acidic stains have negatively charged chromogen which prevents them from penetrating cells because of the negative charge on the surface of bacteria. The background is stained and the cells remain colorless. Heat fixing is NOT used so cells can be seen in their natural shape and size

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gram stain differentiates between

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gram positive and gram negative cells based ondifferences in the chemical composition of cell walls

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gram stain steps

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Primary stain- crystal violet (basic) for 1minuteàboth positive and negative are purple.

Mordant- Gram’s Iodine for 1 minute- helps fixthe dye on or in a cell- both remain purple.

Decolorizing agent- Ethanol (ethyl alcohol) oracetone dropwise until clear- decolorizes gram negative cells but leavespositive cells purple.

Counterstain- Safranin (basic) for 45 seconds-provides a contrast between positive and negative cells- Gram positive arepurple and Gram negative are red/pink

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spore stain

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Spores form to protect against adverseenvironmental conditions and are metabolically inactive; resistant to heat,freezing, staining. Heat must be applied to penetrate the spore. Spores stain green (malachite green) andvegetative cells are decolorized by water and counterstained with Safranin andare red

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acid fast stain

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Diagnostic staining used to identify bacteriathat are not decolorized by acid-alcohol (Mycobacterium tuberculosis, M.leprae). Acid-fast bacteria have a thick, waxy outerlayer high in a lipid called mycolic acid which makes staining more difficult. Heat is needed to stain acid-fast bacteria andacid alcohol doesn’t decolorize it. Carbol fuchsin with heat (to soften the waxycell wall components to facilitate stain penetration)à acid alcohol (acid-fast cellsare not decolorized and remain pink)àmethylene blue (stains non-acid fast cells)· S. aureus are non-acid fast

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selective media

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encourages growth of 1 type of bacteria whileinhibiting growth of another.

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differential media

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distinguishes between 2 or more groups ofbacteria.

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CLED

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differential- distinguishes between lactose fermenters (yellow colonies) and non-lactose fermenters (clear, translucent colonies)

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B-phenyl ethyl alcohol

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selective- inhibits gram negative and allows gram positive cell growth

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MacConkey

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differential and selective- inhibits gram positive growth and selects for gram negative growth; differential for lactose fermenters (bright red colonies)

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Mannitol salt agar

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differential and selective- selective for bacteria that are tolerant for high salt concentrations; differential for mannitol fermentation ability · Positive- yellow colonies

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defined media

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chemically defined or synthetic media; userelatively pure chemicals in known concentrations (you know what and how muchis present); additional vitamins, amino acids, or purines and pyrimidines maybe added

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complex media

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thespecific components and their quantities are not precisely known; made of plantand/or animal extracts which are variable in their chemical composition; it isbased on the organic and inorganic requirements of the microbes; theingredients are in a crude form, prepared by acid digestion or enzymatichydrolysis of materials to release vitamins, minerals, amino acids,polypeptides, and/or carbohydrates

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complex media can include

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Peptone (nitrogen),Yeast extract (B vitamins), Agar (not nutrient, just solidifies the media),Beef extract (salts, vitamins, carbohydrates)

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autoclave

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the use of steam under pressure; increasedpressure raises boiling point of water and produces steam with highertemperature; sterilizes for 15 minutes at 121 degrees C vs. dry heat for 1.5-3hours

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electromagnetic radiations

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UV light does not penetrate but acts as asurface and air disinfectant

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aerobes

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require oxygen, growth at top- B. subtilis andP. aeruginosa

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facultative anaerobes

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grow with or without oxygen; grow best nearsurface, but occur throughout- E.coli

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obligate anaerobe

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require no oxygen; no growth near surface

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aerotolerant anaerobes

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do not use oxygen, but tolerate it; growthevenly throughout tube

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microaerophiles

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reduced oxygen tension; growth just below surface

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catalase test

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tests for ability to produce the enzyme catalase. Hydrogen peroxide added to slant or plate, produces water and oxygen when catalase is present. S. aureus is positive for catalase (bubbles). E. faecalis is negative for catalase. 2 H2O2 -> catalase -> 2H2O + O2(gas)

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Nitrate reduction test

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Reduction of nitrates to supply oxygen to beused as a final electron acceptor during energy formation during anaerobicrespiration. If solution A+B added and becomes pink then nitratereduced to nitrite (E.coli, S. aureus). Negative reaction (no reduction of nitrate):clear after solutions A+B added but turns pink with Zinc (E. faecalis). Nitrate reduced to ammonia or molecularnitrogen: clear after solutions A+B and clear after Zinc (P. aeruginosa)

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extracellular enzyme activities

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Detect exoenzymes that break down largermolecules so they can be transported into cell. Starch agar, milk agar, gelatin hydrolysis

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starch agar

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clear zone of hydrolysis will surround bacterialgrowth if the bacteria produces amylase. B.subtilis

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milk agar

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zone of hydrolysis present if bacterial growthproduces proteases (caseinase). B. subtilis and S. marcescens

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gelatin hydrolysis

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tests for bacteria that produce the enzymegelatinase. Rapid gelatin hydrolysis- remain liquid afterrefrigeration. P. aeruginosa. Slow gelatin hydrolysis- solid afterrefrigeration

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carbohydrate fermentation

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Fermentation- anaerobic process during whichcarbs are broken down for energy production.Positive results: media turns yellow (acidproduction) and gas (air in tube). Facultative anaerobes and aerobes are capable offermentation.

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Triple-sugar iron test (TSI)

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A differential medium that contains lactose, sucrose, glucose, ferrous sulfate, and the pH indicator phenol red. It is used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates. Slant/butt pink=no sugar fermented. Slant pink/butt yellow=glucose only. Both yellow=any of the 3. Butt Black=Hydrogen sulfide produced. Medium pushed up/bubbles=gas production.

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Staphylococcus aureus

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cocci, clusters, gram positive

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Bacillus subtilis

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rod, gram +, spore former

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Escherichia coli

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rod, gram-

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Staphylococcus epidermidis

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cocci, clusters, gram+

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Enterobacter aerogenes

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rod, gram-

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Pseudomonas aeruginosa

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rod, gram-

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Enterococcus faecalis

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cocci, gram+

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Serratia marcescens

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rod, gram-

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Citrobacter freundii

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rod, gram-

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Mycobacteriumsmegmatis

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rod, gram+, acid fast

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Streptococcus pyogenes

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cocci, gram+

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Proteus vulgaris

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rod, gram-

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IMViC Test

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Indole Test- ability to hydrolyze tryptophanwith production of indole. Add Kovac’s reagent- cherry red color indicatesindole is present.

Methyl Red Test- detects presence of largeamounts of acid end products. Add methyl red- red color means acid is present.

Citrate Utilization- ability to ferment citrateand use it as a source of carbon in the absence of glucose. Citrate utilization demonstrated by growth onslant and by blue color change. Contains Bromothymol Blue pH indicator thatturns blue in alkaline conditions

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Hydrogen sulfide test

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motile organisms will produce black colorthroughout; non-motile shows blackening along line of growth. Agar is semi-soft to detect motility

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Wet mounts

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detect motility (E.coli). Brownian movement- cells look like jiggling aswater molecules vibrate, but is not true motility- S. aureus

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Identification of enterics using computerassisted multitest Microsystems

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Used on clinical samples (urine, blood, feces). Advantages: minimal storage space, use lessmedia, rapid results. Disadvantages: difficult to obtain properamounts of inoculum, possible media carryover from one compartment to next,possibility of using nonlog phase culture, relies too much on computer

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Chemical agents of control: disinfectants andantiseptics

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Determines efficacy of antimicrobial agents-affected by concentration, length of exposure, type of bacteria, environmentalconditions (pH, temperature, material). Zone of inhibition- clear zone around disk inwhich no bacteria are growing

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Normal microbiota

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Can cause infection if overgrown. Seen in healthy individuals. Aid in digestion. Prevent infection by opportunistic pathogens.

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a pure culture can be prepared from a mixed slant by

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first separating microorganisms into pure colonies using a steak plate and then growing a pure culture from those colonies

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a colony you isolate is pure if

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it shows a uniform appearance and cultural characteristics

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agar plates are incubated upside down to prevent

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condensation from the lid from creating a wet environment

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plates are incubated 24-48 hours as

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bacteria begin to die after longer than that

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open plates clam style when inoculating to

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limit number of airborne microbes that can contact plate

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total magnification

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ocular lens x objective lens

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when heat fixing

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pass through flame quickly 3 times

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how to negative stain

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1 drop nigrosin on end of slide, aseptically collect small amount bacteria and mix into nigrosin, feather using spreader slide, let air dry

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basic dyes are effective because

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they have a positive charge and are strongly attracted to the negatively charged bacterial cell walls/components

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what does forgetting to heat fix do

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heat fixing binds microorganisms to the slide, so failing to heat fix would result in bacteria washing off slide during staining.

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B-phenyl ethyl alcohol results

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Selects for gram + bacteria. S. Aureus, B. Subtilis were positive and E.coli, P.vulgaris, and E. erogenous were negative

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CLED Results

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Differentiates based on lactose fermentation. E. Coli, S. Aureus, and E. Aerogenes were positive (fermented lactose and made plate yellow). P. Vulgaris was negative.

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Most crucial gram staining step

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decolorizing. Without this all cells would appear purple and indistinguishable.

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You must use fresh cultures with gram stains becasue

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after 24 hours cells begin to die and even gram + cells will stain like gram -

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the purpose of B=phenyl ethyl alcohol in PEA Agar

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is to inhibit growth of gram - and encourage growth of gram+

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how to endospore stain

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Make smear. Heat fix. Primary stain with malachite green over heat for 3-5 min. Rinse with water. Let cool. Counterstain with safranin 30 seconds, rinse with water.

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starch and casein must be

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hydrolyzed outside the cell as they are too large to pass through the cell membrane

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enzymes in starch hydrolysis

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alpha-amylase, beta-amylase, alpha-1,6-glucosidase

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enzyme in casein hydrolysis

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casease

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necessary to add iodine to starch hydrolysis because

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to visualize starch and its sugar subunits by staining it brown, i.e., starch hydrolysis is indicated by a clear zone surrounding growth

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results of starch hydrolysis

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monosaccharide alpha-glucose, disaccharide alpha-maltose, beta-maltose

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We mostly work with

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gram- rods