Lab Exam Flashcards
Principle of the Methyl Red test
Assuming that mixed acid fermentation is the primary energy source for a bacteria when this occurs the pH drops drastically below 5. pH greater than 5 causes the methyl red to turn yellow
How to fill the Rapid test capules
Fill Fully-CIT, VP,GEL
Fill half way- All remaining tubes
Add Parafin oil-LDC, ODC,ADH,H2S, URE
Gram Stain scoring
Purple-> Gram positive
Pink->Gram negative
How to perform the catalase test
1)Add Hydrogen peroxide to a slide and bacteria loop into it
2)Observe for bubbles
Enriched media
Media which adds one or more nutritional supplements to a medium allowing a fastidious organism growth to increase
Oil Immersion microscopy
Uses oil to decease the diffraction from the air to glass surface with an oil that has the same defractive index as the microscope. 100x objective
MacConkeys agar
is a selective and differential culture medium for bacteria. It is designed to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacteria and differentiate them based on lactose fermentation.Lactose fermenters turn red or pink on MacConkey agar, and nonfermenters do not change color. Inhibits gram positive bacteria.
Principle of the KOH test
KOH will dissolve the thin peptidoglycan layer of G- bacteria but will not affect the G+ bacteria. The stickiness is a result of the bacterial DNA strands being released
How to prepare an unstained mount
1)Place one drop of infusion onto a blank slide
2)Add a cover slip
3)Begin by looking for the edge of the cover slip on 10x objective on bright field settings
4)Switch to phase contrast on the 40x objective
Phase contrast Microscopy
Allows for viewing of unstained and living specimens, by generating constructive interference between scattered and background light rays in regions of the field of view that contain the specimen, and by reducing the amount of background light that reaches the image plane, 40x objective
Motility observation
Prepare a wet mount of the bacteria and observe under 40x zoom with phase contrast and observe motility.
Kohler Illumination
Evenly illuminates the field of view, provides a clear image without reflection or glare and minimizes heat on a specimen. Done on 10x magnification
How to perform the fermentation of carbohydrates test
1)Inoculate the tube of phenol red carbohydrates broth with an organsim
2)Leave over night and score for Acid, gas, and growth
How to score the Catalase test
Positive for Catalse-> bumbles in H2O2
Negative for Catalase->no bubbles in H2O2
How to score the Indole Production test
Red Alcohol layer- Indole positive
Clear Alchol layer- Indole negative
How to score the Citrate Utilization test
No growth- NG
Yellow-acidic and positive
Blue-Alkaline and positive
NO colour change- Negative
Bacterial Elevation morphology
1)Flat
2)Raised
3)Convex
4)Pulvinate
5)Umbonate
What does PCR do
is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA
Principle of the RYU flagella stain
The Flagella Stain provides
a method for viewing bacterial flagella by employing crystal violet in an alcoholic solution as the primary stain.
During the staining procedure, the alcoholic solution evaporates and leaves a precipitate around the flagella,
increasing its apparent size to the human eye.
How to perform aseptic technique
1) When taking out inoloops do not let them touch
2)Flame the mouth of the incoulum before and after use
3)Work next to an open flame
4)Do not let the tip of the inoloop touch the sides of the glass
How to perform and score the starch hydrolosis test
1)Perform a streak plate onto Starch agar
2)Dump Iodine on the plate if a halo forms then it is starch positive, if no halo present then it is starch negative
How to perform the Voges-Proskauer Test
1)Inoculate Mr-VP broth and incubate for 24 hours
2)Add 1ml of culture into a test tube under fume hood and add 0.5ml of Barrits reagent A and 0.5 ml Barrits reagent B, mix for 30 seconds
3)Look for Pink colour to develop into Red
How to perform the Gelatinase test
1)Innoculate bacteria into gelatin broth and incubate overnight
2)Vortex the tubes and place tubes in a beaker for 5 minutes
3)Check to see if the gelatin solidifies
Azide blood agar
Used for the selective isolation and cultivation of Staphylococcus and Streptococcus species from mixed bacterial flora. Inhibits growth of Gram negative bacteria
Principle behind the gelitanse test
Gelatin is a liquid at 37C but solidifies on ice, if gelatin hydrolysis has occurred then the gelatin will not solidify. Geltinase catalyses the hydrolysis of gelatin into peptides and carbon source.
How to perform and score the latex agglutiation test
Add antigen and antiserum to a latex agglutination card. If positive it will form granules indicating reaction of antigen and antibody
How to score the oxidase test
Positive for Cytochrome oxidase- Purple stain
Negative for Cytochrome oxidase- No change in filter paper colour
Mannitol salt agar
used as a selective media for the isolation of pathogenic Staphylococc, it contains a high concentration (about 7.5–10%) of salt which is inhibitory to most bacteria - making MSA selective against most Gram-negative and selective for some Gram-positive bacteria. Also a diffrential media If an organism can ferment mannitol, an acidic byproduct is formed that causes the phenol red in the agar to turn yellow.
Tryptic soy broth
BD Tryptic Soy Broth (Soybean-Casein Digest Medium) is a general purpose liquid enrichment medium used in qualitative procedures for the sterility test and for the enrichment and cultivation of aerobic microorganisms that are not excessively fastidious.
Bacterial whole colony morpholgies
1)Punctiform
2)Circular
3)Filamentous
4)Irregular
5)Rhizoid
6)Spindle
m-ENDO agar
Coliforms give a dark red colour of a metallic sheen. Clear are non coliform colonies
How to perform the Methyl Rest test for Mixed acid fermentation
1)Use the MR-VP solution as the culture for this test
2)Add Methyl red to the culture, if yellow no fermentation, red = fermentation
Differential media
Incorporates reagents or chemicals that permit the observer to differential between different types of bacteria after they have grown through colour or colony morphology ex.Blood agar and hemolytic bacteria
How to perform the oxidase test
TMPD filter paper is touched to an organism to determine presence of Cytochrome oxidase leading to a colour change of black or purple
Plate count technique
1)Perform a serial dilution based on the OD600 reading of the bacteria to reduce the #of cells/ ml
2)Perform a spread plate of 0.1ml of diluted substance
3)Count # of colonies and use this to determine the original concentration of bacteria
How to perform the Gram stain
1) Stain with Crystal violet for 60 seconds, rinse and pat down with paper
2)Flood with grams iodine, wait 60 seconds, rinse and pat
3)Decolonize with alcohol for 10-20 seconds
4)Counter stain with safranin for 10-20 seconds blot off water
5)air dry
Principle of the endospore stain
Green is retained by the endospore due to the thick coating. By steaming the smear it allows the dye to penetrate the coating and stay there after decolourizing
Principle behind the catalase test
Catlase is an enzyme that decomposes H2O2 into water releasing gas, aerobic bacteria possess catalase as it removes H2O2 when performing the oxidation reduction. The gas produced from the Catalase test indicates the presence of the enzyme as it reacts with the H2O2 on the slide
How to score the Voges-Proskauer test
Red-Positive for Acetoin
Pink- Negative for Acetoin
Principle of the Citrate Utilization test
Use of citrate as an energy source causes the pH to increase (8.4) causing a blue colour or a decrease causing a yellow colour (6.8). If no reaction occurs the medium stays green
List the bacterial flagella shapes
1)Polar-single flagella
2)Lophotrichious- tufts of flagella on one pole
3)Amphitrichious- Flagella on either side
4)Peritrichious- flagella all over
How to score the Methyl Red test
Yellow=No mixed acid fermentation
Red=Mixed acid fermentation has occured
Principle of the Indole Production test
Indole is produced when Tryptohan is broken down with Tryphtophanase, the indole byproduct will react with Kovac’s reagent to produce a red layer. When a carbohydrate source available no tryptophan is used and idole will not be produced.
Semi-solid agar motility assay
Determines the motility of bacterial species by suspending it in a semi solid agar that an organism can swim through. The spread of organism in a radial shape through the agar indicates motility of organism.
Principle of the Voges Proskauer test
Acetoin is an intermediate product in butanediol fermentation and is detected when reacted with Barrits reagents
Optical density reading
1)Put blank into the slot
2)Click calibrate by clicking on the gran menu
3)Put sample in and click collect for 5-10 seconds
4)Scroll to the wavelength on intrest and read the optical density
5)Repeat with other samples
6)Increasing OD600 reading means increasing bacterial density as there is less light that can pass through the sample.
How to perform the Endospore stain
1)Prepare a smear of culture on a slide
2)Place filter paper on the slide and saturate with malachite green
3)Heat the bacteria over hotplate for 5 minutes to fix
4)Dispose of filter and rinse with water
5)Counter stain with safranin
6)blot with water and dry
How to score the fermentation of carbohydrates test
Acid- Red colour
Basic-Yellow colour
Gas-Fermentation
No gas-No fermentation
How to perform a spread plate
1) Use alcohol and burn the spreader
2)Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petridish underneath at the same time.
How to score the culture tube agglutination test
Score the degree of agglutination by the presence of white percipitate at the bottom of the tube. The concentration of antiserum corresponding with a clumping reaction is the titer of antiserum calculated by inverting the dilution factor.
IMViC test
I=Indole
M=Methyl Red
V=Voges Proskaur
C=Citrate
Used to identify Enterobacteriaceae, gram negative,oxidase negative,faculative anerobic rods
How to score the gelatinase test
Tube solidifies-Negative gelatin reaction
Tube remains liquid- Positive gelatin reaction
Principle behind the Gram stain
1)Purple colour will stain gram positives thick cell wall
2)Iodine sets the colour into the cell wall
3)Alcohol removes excess stain
4)Saffron will stain the clear gram negative bacteria
Principles behind the nitrate Reductase test
Nitrate acts as a terminal electron acceptor in anaerobic respiration so the presence of nitrate reduction indicates that it is used as an electron aceptor. Some organisims further reduce this nitrite and reacting the solution with Zinc will indicate that this has occured
How does ELISA work
In a direct ELISA, the antigen is bound to the bottom of the microplate well, and then it is bound by an antibody that is specific to the antigen and also conjugated to an enzyme or other molecule that enables detection. By using an ELISA concentration curve a standard curve can be built and a linear formula found to determine concentration of an unknown anitbody
How does Agarose Gel Electrophoresis work
the molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths.
Nutrient Broth
Pre-enrichment in a nonselective medium such as Nutrient Broth allows for cell damage repair, dilutes toxic or inhibitory substances, and provides a nutritional advantage to Salmonella over other bacteria.
Kirby Bauer Disk Method
1)Create a spread plate on Mueller Hinton agar with 0.1ml of inocculum
2)Add antibiotic disks onto the plate and incubate over night
3)Measure the zone of inhibition and compare to a data table to score the sensitivity from resistant, intermediate or susceptible
Unit size for 10x, 40x, and 100x microscopy
10x= Ocular unit(10dashes) 100um
40x=Ocular units 25um
100x=10um
MIC Test
1)Serially dilute the antibiotic six times in mueller hinton broth
2)Incubate overnight
3)Find the lowest concentration of antibiotic that results in no growth
Levine EMB agar
A selective medium because it inhibits Gram-positive bacterial growth. The two dyes in this medium, eosin and methylene blue, act as selective agents to inhibit Gram-positive bacteria, but allow for the growth of Gram-negative bacteria. Organisms that ferment lactose appear dark/black or green often with “nucleated colonies”—colonies with dark centers.[3] Organisms that do not ferment lactose will appear pink and often mucoid.
How to perform the triple sugar iron agar test
Stab the inoneedle into a deep agar and slide over the slant of the Triple sugar iron agar
Bacterial Margin morphology
1)Entire
2)Undulated
3)Lobate
4)Erose
5)Filamentous
6)Curled
How to perform a streak plate
1) Divide the plate into 4 sectors
2) Using innoculum completely fill one area with high concentration of bacteria
3)Use an inoloop and touch the spread area and pull into the next sector
4)Repeat 4 times to get individual colonies
How to interpret the results of the endospore stain
Green->Endospores
Red->Regular cell
Principle behind the oxidase test
Aerobic bacteria use Cytochrome oxidase as the final electron acceptor in the oxidative phosphorylation chain. The oxidase test determines if a bacteria uses this particular pathway by looking for the final electron acceptor
How to score the KOH test
Nonsticky, Negative result->gram positive
Sticky, Positive result->gram negative
Principle behind the starch hydrolosis test
Bacteria capable of producing extracellular amylase enzymes can hydrolyze the starch and produce smaller water-soluble sugar molecules like glucose, maltose, and dextrose. Iodine reacts with unused starch hydrolysis of the starch will create a clear zone around the bacterial growth.
How to score the reduction of nitrates by nitrate reductase test
Red with Sulphanic acid and dimethyl-alpha-naphthylamine, nitrate recution into N0,N2O, or N2
Red with addition of zinc- Reduction of nitrates further than NO,N2O, or N2
No reaction- No reduction of nitrates
Principle behind the triple sugar iron agar test
Tests for gram negative bacteria ability to ferment glucose, lactose and sucrose as well as ability to produce Hydrogen sulphide gas
How to score the Immunodiffusion Ouchterlony plate
Formation of white band indicates the interaction of antibody with antigen. If the white bands overlap this indicates similar antigenic components between antigens
Glucose mineral salt broth
All necessary ions plus glucose
Principles behind the Fermentation of carbohydrates test
Red colour indicates use of the energy source
Gas bubble indicates the byproducts of fermentation
How to preform the RYU flagella stain
1)Add water to a microscope slide and add innoculum
2)Place cover slip on top and let sit 5-10 minutes
3)Apply flagella stain at the edge of the coverslip ad wait 5-10 minutes
4)View under oil immersion
Describe how to use a hemocytometer
1)Set up microscope of Kohler illumination
2)Place a hemocytometer and cover slip on the stage and focus on the grid
3)add methylne blue to the cell suspension and add a small drop of culture to the chamber
4)Count cells that are not dyed blue and then divide by the volume of the chamber for #of viable cells/mL
How to prepare and score a bacteriophage plate
1)Mix bacteriphages with an excess of chosen bacteria
2)Suspend the solution in molten agar
3)Pour the solution onto a plate and incubate
4)Count the number of clear colonies on the plate to determine number of bacteriophages
How to perform the reduction of nitrates by nitrate reductase test
1)Inoculate trypticase nitrate broth and incubate for 48 hours
2) Add 1ml of sulphanic acid and 1ml of dimethyl-alpha-naphthylamine to the tubes
3)Red or maroon is positive, no reaction proceed to next step
4)Add zinc powder and see if reaction occurs
How to perform the Indole Production Test
1)Inoculate trypticase broth/phytone and incubate for 48 hours
2)Add 1ml of Kovac’s Solution
3)Look for reddening of alcohol layer for indole
How does MALDI-TOF work
an analytical technique in which particles are ionized, separated according to their mass-to-charge ratio, and measured by determining the time it takes for the ions to travel to a detector at the end of a time-of-flight tube. The specific pattern is compared to a database to provide the identity of an unknown organism
Semi enriched broth
Glucose mineral salt broth plus 1/10th nutrient broth
Selective media
Incorporates one or more reagents which inhibit the growth of certain groups of bacteria and have a minimal effect on growth rate of others. Ex.crystal violet inhibits gram positive bacteria
How to score the triple sugar iron agar test
Alkaline slant (pink)/ Acid Butt (yellow)-Glucose is fermented
Acid slant(yellow)/Acid butt(yellow)-Glucose and/or lactose, sucrose is fermented
Bubbles or cracks- Gas is produced
Black precipitate- Hydrogen sulphide gas is produced
Agar deep
Allows for observation of the oxygen requirements of microbes, allowing you to determine if the organism is an anaerobe, aerobe, of faculative anaerobe
How to perform the citrate utilization test
1)Inoculate Agar slant of Simmons citrate medium by stab and surface streak
2)Incubate 24 hours
3)Record growth as NG, A, K, -
Thioglycolate tube
1: Obligate aerobes need oxygen because they cannot ferment or respire anaerobically. They gather at the top of the tube where the oxygen concentration is highest.
2: Obligate anaerobes are poisoned by oxygen, so they gather at the bottom of the tube where the oxygen concentration is lowest.
3: Facultative anaerobes can grow with or without oxygen because they can metabolise energy aerobically or anaerobically. They gather mostly at the top because aerobic respiration generates more ATP than either fermentation or anaerobic respiration.
4: Microaerophiles need oxygen because they cannot ferment or respire anaerobically. However, they are poisoned by high concentrations of oxygen. They gather in the upper part of the test tube, but not the very top.
5: Aerotolerant organisms do not require oxygen as they metabolise energy anaerobically. Unlike obligate anaerobes, though, they are not poisoned by oxygen. They can be found evenly spread throughout the test tube.
