Lab Exam Review Sheet

Biology 271 lab Study Guide with hints
Winter 2011

Note: Location and use of all laboratory equipment (ex. Autoclave, incubators, loops, needles ,disposal containers, staining equipment, burners, thermal cycler…)

1. Use and care of microscope and special adjustments. Be able to label parts and describe their use.
o Type of microscope used in class (light microscope)
o Iris diaphragm (controls amount of light entering condenser)
o Magnification (enlargement of image)
o Resolution
o Which objective is the oil immersion objective (100X)
o What is the purpose of using immersion oil
o What is the diopter adjustment for (adjusts for difference in eye strength)
o Interpupilary distance

2. What is a pure culture (culture containing progeny of one bacterial cell)

3. Describe the steps performed when using Aseptic Technique (you should know the steps)
o What is the purpose of using Aseptic Technique

4. What is a streak plate; what is its purpose (obtaining pure cultures; separating bacteria). What is a lawn ( thin layer of bacteria covering surface of Petri dish); what is its purpose (antibiotic disc sensitivity[Kirby Bauer methoth] testing, disc dilution testing for disinfectants).

5. Be able to describe the use of the following media:
o EMB agar
o Mannitol Salts Agar (remember, for Staph- high salt content)
o Simmons Citrate Agar (remember Enterobacter versus E. coli growth)
o TSA (general purpose growth media for fastidious bacteria)
o Mueller Hinton Agar(used for antibiotic disc sensitivity testing)
o TSA + Sheeps blood (hemolysis)

6. Describe the use and theory behind the following:
o Thioglycolate (remember, sodium thioglycolate removes oxygen from media)
o Stab
o Shake

7. What does a zone of inhibition indicate in Antibiotic sensitivity testing

8. Describe the structure of the pGlo plasmid
o What is the purpose of arabinose used in this experiment (switch)
o What is the purpose of the antibiotic used in this experiment (selection)
o Which characteristic indicated transformation? (growth in presence of antibiotic (ampicillin))

9. What is the Biolog Identification system (To be completed)
o List the steps used to perform the biology identification ( grow bacteria according to specifications to ensure optimum metabolic activity, prepare
bacteria concentration using standard, incubate for 24 hours at proper temp., read plate)
o What is the SIM index (indicates likelihood of identification based on relatedness of cluster of identified bacteria)
o What does probability indicate (likelihood of correct identification)
o Why is it important to calibrate your transmittance meter with the correct standard (to ensure correct concentration of bacterial solution)
o Why is thioglycolate added to some bacterial suspensions (prevents capsule production)

10. What is the Enterotube test system- how is it used
o Which type of organisms can be used with this test system (Enterobacteriaceae)
o Which test(s) should you perform prior to using this system (oxidase)
o Can this test system be used with a broth culture? (best to use cultures grown on solid media – Petri dish)
o Can gas production be detected using this system (yes, remember glucose compartment)

11. DNA extraction: Describe the spooling technique for extracting DNA from Ecoli. (remember EDTA, sarkosyl, RNAase, alcohol…) Why is it important to be ‘gentle’ when performing this procedure.
o What were the reagents(remember EDTA, sarkosyl, RNAase, alcohol…)? What was their purpose?

12. Describe Agarose Gel electrophoresis- understand principles and technique
o What type of gel electrophoresis did we perform in lab (horizontal or vertical).
o What is the sample to be tested usually mixed with (what is a tracking dye; why is it used)- to weigh the sample down – so that it sinks into the wells and to allow visualization of DNA as it moves on the gel)

13. Alu-Human DNA Typing (PCR experiment)
o What are introns
o What are polymorphisms? How are they used in Human DNA typing
o What is PCR? (briefly describe steps), What is a Thermal Cycler?
o In general, what step were used to prepare DNA for electrophoresis during the Alu-Human DNA typing (ex. Islolated sample cell material, lyse cells to extract DNA, Amplify DNA via PCR… What were the reagents and what were they used for?)

14. Describing the following staining procedures in detail, including all reagents
o Gram Stain

15. Slide set (as indicated in lab… Gram neg vs. Gram positive cells cell shape, capsule stain, flagella stain, negative stain, acid fast stain, spiral forms)

16. Laboratory safety rules

17. ELISA – understand the principles and technique
o What is the purpose of the washing steps
o Why is a secondary Ab used?
o What enzyme system did we use? What color indicated positive test?

18. What is Alfa, beta and gamma hemolysis. How was this demonstrated in lab.

19. What is a bacticinerator?

20. What is the usual time/temperature achieved in an autoclave for sterilizing lab glassware. (use C for temp)? ---(121C at 15lbs/inch for 15 minutes)

21. What is room temperature (in C)? about 25C

22. What are the procedure and reagents and for each of the following test, and indicate what enzyme or product was being detected, know what are positive and negative tests results:
o Catalase test (hydrogen peroxide-look for bubbles)
o Oxidase test (dry slide oxidase test – purple color within 20 seconds)
o Gelatin hydrolysis (use gelatin media, gelatin cannot revert to solid state)
o IMVic test (Indole, methyl red, VP test – used to differeniate enterobacteria based on acid production and fermentation products---see lab handouts)
o Carbohydrate fermentation
o Urea hydrolysis

Use and care of microscope and special adjustments. Be able to label parts and describe their use.
o Type of microscope used in class (light microscope)
o Iris diaphragm (controls amount of light entering condenser)
o Magnification (enlargement of image)- the ability of the microscope to increase specimen size
o Resolution-the ability of the microscope to reveal specimen structure
o Which objective is the oil immersion objective (100X)
o What is the purpose of using immersion oil-
oil with a density more akin to the microscope lens that of water helps to decrease the loss of transmitted light, which, in turn, increases image clarity
o What is the diopter adjustment for (adjusts for difference in eye strength)
o Interpupilary distance-the distance between the center of the pupils of the two eyes

2. What is a pure culture (culture containing progeny of one bacterial cell)
a laboratory culture containing a single species of organism.


3. Describe the steps performed when using Aseptic Technique
a.incenerate llop
b. uncap tube and pass in front of incenorator
c. put loop in tube
d. sweep loop across agar plate
incenerate loop
pass tube in front of incenorator
cap tube
o What is the purpose of using Aseptic Technique- to prevent contamination of your culture with organisms from the environment and to prevent the culture from contaminating you or others.

4. What is a streak plate- a plate of agar used to incubate a culture
what is its purpose? -obtaining pure cultures; separating bacteria
What is a lawn- thin layer of bacteria covering surface of Petri dish;
what is its purpose -antibiotic disc sensitivity[Kirby Bauer methoth] testing, disc dilution testing for disinfectants

5. Be able to describe the use of the following media:
o EMB agar-Used to detect gram negative, enteric bacteria and differeniate nonlactose fermenters from coliforms.
o Mannitol Salts Agar (remember, for Staph- high salt content)-Use for isolating Staphylococci. Staphylococci are not inhibited by a concentration of 7.5% sodium chloride (most other pathogenic bacteria are). Phenol Red is the pH indicator; mannitol is the carbohydrate source. Fermentation of mannitol lower the pH of the media causing the phenol red to turn yellow in color. Only pathogenic Staphylococcus ferment mannitol.
o Simmons Citrate Agar (remember Enterobacter versus E. coli growth)-Used to differentiate Enterobacteriaceae, specifically, the fecal coliforms from non-fecal coliforms). Fecal coliforms (such as E. coli) are not able to use citrate as the sole carbon source in the media. Nor are they able to use inorganic ammonium salt as their sole nitrogen source. Non-fecal coliforms (such as Enterobacter aerogenes or Salmonella enteritidis) can grow in a media that contains citrate as the carbon source and ammonium salts as the nitrogen source. Brom thymol blue has been added to the media to test for alkaline products of metabolism.
o TSA (general purpose growth media for fastidious bacteria)
o Mueller Hinton Agar(used for antibiotic disc sensitivity testing)
o TSA + Sheeps blood (hemolysis)


6. Describe the use and theory behind the following:
o Thioglycolate (remember, sodium thioglycolate removes oxygen from media)-Sodium thioglycollate binds free oxygen. This media contains a redox potential indicator (an indicator which detects the presence of oxygen)
o Stab-Deeps are used to grow anaerobic or facultative bacteria. Deeps are inoculated using the stab technique
o Shake- helps to maintain an anaerobic environment

7. What does a zone of inhibition indicate in Antibiotic sensitivity testing-It is the area on an agar plate where growth of a control organism is prevented by an antibiotic usually placed on the agar surface. If the test organism is susceptable to the antibiotic, it will not grow where the antibioitic is.


8. Describe the structure of the pGlo plasmid-
pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows:
-Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family)
-araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose)
-GFP, the green fluorescent protein

Like most other circular plasmids, the pGLO plasmid contains an origin (ori), which is a region of the plasmid where replication will originate.

o What is the purpose of arabinose used in this experiment (switch)- initiates transcription of the GFP gene resulting in fluorecent green cells under UV light
o What is the purpose of the antibiotic used in this experiment (selection)- to select bacteria containing the DNA of interest
o Which characteristic indicated transformation? -growth in presence of antibiotic (ampicillin)

10. What is the Enterotube test system- how is it used-
The Enterotube is a multitest system to detect enteric pathogens. Its identification depends on the metabolic activity of the pathogen.
Which type of organisms can be used with this test system (Enterobacteriaceae)
o Which test(s) should you perform prior to using this system (oxidase)
o Can this test system be used with a broth culture? (best to use cultures grown on solid media – Petri dish)
o Can gas production be detected using this system (yes, remember glucose compartment)

Biology 271 lab Study Guide with hints
1. Use and care of microscope and special adjustments. Be able to label parts and describe their use.
• Type of microscope used in class (light microscope)
• Iris diaphragm (controls amount of light entering condenser)
• Magnification (enlargement of image)- the ability of the microscope to increase specimen size
• Resolution-the ability of the microscope to reveal specimen structure
• Which objective is the oil immersion objective (100X)
• What is the purpose of using immersion oil- oil with a density more akin to the microscope lens that of water helps to decrease the loss of transmitted light, which, in turn, increases image clarity
• What is the diopter adjustment for (adjusts for difference in eye strength)
• Interpupilary distance-the distance between the center of the pupils of the two eyes
2. What is a pure culture- (culture containing progeny of one bacterial cell) a laboratory culture containing a single species of organism.
3. Describe the 7 steps performed when using Aseptic Technique
a. incinerate loop
b. uncap tube and pass in front of incinerator
c. put loop in tube
d. sweep loop across agar plate
e. incinerate loop
f. pass tube in front of incinerator
g. cap tube
What is the purpose of using Aseptic Technique- to prevent contamination of your culture with organisms from the environment and to prevent the culture from contaminating you or others.
4. What is a streak plate- a plate of agar used to incubate a culture; what is its purpose? -obtaining pure cultures; separating bacteria
What is a lawn- thin layer of bacteria covering surface of Petri dish; what is its purpose -antibiotic disc sensitivity [Kirby Bauer method] testing, disc dilution testing for disinfectants
5. Be able to describe the use of the following media:
• EMB agar-Used to detect gram negative, enteric bacteria and differentiate nonlactose fermenters from coliforms
• Mannitol Salts Agar (remember, for Staph- high salt content)-Use for isolating Staphylococci. Staphylococci are not inhibited by a concentration of 7.5% sodium chloride (most other pathogenic bacteria are). Phenol Red is the pH indicator; mannitol is the carbohydrate source. Fermentation of mannitol lowers the pH of the media causing the phenol red to turn yellow in color. Only pathogenic Staphylococcus ferment mannitol
• Simmons Citrate Agar (remember Enterobacter versus E. coli growth)-Used to differentiate Enterobacteriaceae, specifically, the fecal coliforms from non-fecal coliforms). Fecal coliforms (such as E. coli) are not able to use citrate as the sole carbon source in the media. Nor are they able to use inorganic ammonium salt as their sole nitrogen source. Non-fecal coliforms (such as Enterobacter aerogenes or Salmonella enteritidis) can grow in a media that contains citrate as the carbon source and ammonium salts as the nitrogen source. Brom thymol blue has been added to the media to test for alkaline products of metabolism
• TSA (general purpose growth media for fastidious bacteria)
• Mueller Hinton Agar(used for antibiotic disc sensitivity testing)
• TSA + Sheep’s blood (hemolysis)
6. Describe the use and theory behind the following:
• Thioglycolate (remember, sodium thioglycolate removes oxygen from media)-Sodium thioglycollate binds free oxygen. This media contains a redox potential indicator (an indicator which detects the presence of oxygen)
• Stab-Deeps are used to grow anaerobic or facultative bacteria. Deeps are inoculated using the stab technique
• Shake- helps to maintain an anaerobic environment
7. What does a zone of inhibition indicate in Antibiotic sensitivity testing-It is the area on an agar plate where growth of a control organism is prevented by an antibiotic usually placed on the agar surface. If the test organism is susceptible to the antibiotic, it will not grow where the antibiotic is.
8. Describe the structure of the pGlo plasmid-
pGLO is made up of three genes that are joined together using recombinant DNA technology. They are as follows:
-Bla, which codes for the enzyme beta-lactamase giving the transformed bacteria resistance to the beta-lactam family of antibiotics (such as of the penicillin family)
-araC, a promoter region that regulates the expression of GFP (specifically, the GFP gene will be expressed only in the presence of arabinose)
-GFP, the green fluorescent protein
Like most other circular plasmids, the pGLO plasmid contains an origin (ori), which is a region of the plasmid where replication will originate.
• What is the purpose of arabinose used in this experiment (switch)- initiates transcription of the GFP gene resulting in fluorescent green cells under UV light
• What is the purpose of the antibiotic used in this experiment (selection)- to select bacteria containing the DNA of interest
• Which characteristic indicated transformation? -growth in presence of antibiotic (ampicillin)
10. What is the Enterotube test system- how is it used- The Enterotube is a multi-test system to detect enteric pathogens. Its identification depends on the metabolic activity of the pathogen.
Which type of organisms can be used with this test system (Enterobacteriaceae)
Which test(s) should you perform prior to using this system (oxidase)
Can this test system be used with a broth culture? (Best to use cultures grown on solid media – Petri dish)
Can gas production be detected using this system (yes, remember glucose compartment)

11. DNA extraction: Describe the spooling technique for extracting DNA from Ecoli. (remember EDTA, sarkosyl, RNAase, alcohol…)
Isolating DNA:
1. Add 2 ml of EDTA Buffer to a tube of 5 ml of resuspended cells
2. Add .5 ml of RNase solution to digest the RNA, tightly cap and invert the tube several times
3. Incubate for 5 minutes at room temp
4. Add .5 ml of Sarkosyl solution and 1 ml of protease solution to break up the cell wall. Cap and invert 3 times. The protease solution will further cut up intracellular proteins, especially the folded ones.
5. Incubate for 20 minutes in a 45 degree water bath
6. Add 0.6 ml of 5M NaCI , invert several times (this prepares DNA almost to the point of coming out of the solution)—slowly pour into a clean 50 ml beaker and proceed to spooling technique


Spooling DNA:
1. First overlay the viscous DNA solution with ice cold alcohol
2. Submerge a glass rod just below the interface of the alcohol and the DNA solution and twirl and swirl the rod several times to spool the DNA onto the rod
3. Remove the rod and verify that you have a gelatinous looking translucent precipitate collecting on the end
4. Continue to the steps to re-dissolve the DNA technique, when you have collected all that you can on the rod
Why is it important to be ‘gentle’ when performing this procedure? When you spool the DNA you do not want to shear it into too many different sizes or it will not appear as bands when you electrophorese it, rather it will appear as a smear and not be legible.
What were the reagents (remember EDTA, sarkosyl, RNAase, alcohol…)? What was their purpose?
-EDTA – to chelate, or “form complexes with sever metal ions”
-sarkosyl—Dissolves cell membrane and denatures many proteins
- RNAase—a proteolytic enzyme is added to the solution to digest all proteins that are free in the solution and bound to the DNA
-alcohol—added to create an interface between the two liquids in the experiment (the DNA with a higher molecular weight, will be caused to sink to the bottom of the interface- thus separating it to spool it)
Describe Agarose Gel electrophoresis- understand principles and technique
- Used for analyzing DNA in this size range (?).
- Gel consists of microscopic pores that act like a molecular sieve
- The gel is placed into a buffered solution inside the electrophoresis chamber
-The gel is cast with wells in it and the DNA is loaded into these wells after having a loading gel added to it for weighting the DNA
-The direct current is applied, and forces the strongly negative charged DNA (due to the neutral buffer solution) through the pores in the gel toward the positive electrode according to the size of the DNA fragments—the smaller the fragment, the faster it will migrate through the gels pores
- NOTE: if the DNA is badly sheared and there are too many sizes of fragments it will appear as a smear after done- not as bands-this is why the spooling technique is done gently
What type of gel electrophoresis did we perform in lab (horizontal or vertical)? Horizontal
What is the sample to be tested usually mixed with (what is a tracking dye; why is it used) - to weigh the sample down – so that it sinks into the wells and to allow visualization of DNA as it moves on the gel)
Alu-Human DNA Typing (PCR experiment)
What are introns? Non coding DNA within the genes of Eukaryotes
What are polymorphisms? How are they used in Human DNA typing –is the occurrence of multiple allele at a locus (the location of the gene on the chromosome) of recent random mutations. For example, there are 95, 207, and 50 different alleles at the HLA-A, HLA-B and HLC loci, respectively. Thus within the human species, there are multiple polymorphisms at each locus. However each individual has only two of these alleles at each locus (one allele is of paternal origin and one is of maternal origin.)
What is PCR? (Briefly describe steps)- it is a technique used to produce exponentially large amounts of a specific piece of DNA from a template (small amount of material). “Amplify DNA”
Steps:
1. Denaturation (at 94c-double strand of DNA melts open to a single strand of DNA- 1 min
2. Annealing at 54◦C –bonds are constantly formed and broken between the single stranded primer and the single stranded template- 45 sec forward and reverse primers
3. Extension At 72◦C -ideal working temperature for the polymerase, primers that are on positions with no exact match/ get loose again due to the high temp and don’t give an extension of the fragment- 2 min only dNTP’s.
30-40 cycles of the three steps.

What is a Thermal Cycler? The thermal cycler (also known as a Thermo cycler, PCR Machine or DNA Amplifier) is a laboratory apparatus used to amplify segments of DNA via the polymerase chain reaction (PCR) process. The device has a thermal block with holes where tubes holding the PCR reaction mixtures can be inserted. The polymerase chain reaction is carried out repeatedly in cycles of different temperatures for specific times in the thermo cycler

In general, what steps were used to prepare DNA for electrophoresis during the Alu-Human DNA typing (ex. Islolated sample cell material, lyse cells to extract DNA, Amplify DNA via PCR… What were the reagents and what were they used for?)
Steps:
1. Denaturation at 94◦C
2. Annealing the primers to the separated DNA template strands through rapid cooling in a thermo cycler to 60◦C
3. Extension at 72◦C of the Taq DNA polymerase to make complete copies of each template DNA strand
Note: Denaturation-Annealing-Extension=1 complete thermal cycle

Describing the following staining procedures in detail, including all reagents:

Gram neg cells—thin peptidoglycan layer- stains pink
vs.
Gram positive cells—Thick peptidoglycan layer-stain purple
Gram Stain (30/60/5/30)—Steps: Prepare smear/heat fix/cover with violet stain for 30 seconds/rinse and flood with iodine for 60 sec/rinse and flood with decolorizer for 5 seconds (this removes crystal dye-iodine complex from gram neg)/rinse and flood with saffrinin for 30 seconds/Rinse and blot/clean back with alcohol swab or wipe
cell shape—Negative = thin peptidoglycan layer/Positive= Thick peptidoglycan layer
Simple Stain—A procedure using one stain.
Steps: Apply an even thin layer of preparation (liquid) to the slide and allow to air dry for 5-10 minutes/heat fix by passing 3 times over bacticinerator. Cover slide with crystal violet stain/wait 20 seconds and rinse with tap water/blot dry gently as to not disturb the cells.
Capsule stain—Wet mount- used for capsule staining due to poor staining ability. Steps: Make suspension of an organism in a drop of water on a clean slide/Put a drop of India ink next to it/carefully lower a coverslip over the two drops so that they mix together/ There should be a gradient in the concentration of the ink/Examine under microscope and find a field where you can see the cells surrounded by a halo in a black background/bacteria are not killed in the staining process, so boil slides in a beaker of boiling water for 2-3 minutes before cleaning
flagella stain—Where we observed pre-stained slides that were built up with mordant (such as iodine) prior to us viewing them. Too difficult to do.
negative stain—A simple stain in which the organisms appear clear against a dark background (think negative photograph). Mix one loopfuls of organisms and one loopfuls of India ink together on a slide/use a clean slide to create a thin smear on the slide/allow to dry-heat fix 10 passes over bacticinerator and observe under microscope.
acid fast stain—Useful for identification of bacteria with a waxy lipid cell wall (mostly mycobacteria). These are all gram positive, but lipid in cell wall prevents gram-staining.
Steps: Prepare a smear and heat fix/ Cover smear with Kinyoun carbolfucsin and stain for 3-5 minutes-no heat/Rinse with H2O/decolorize with acid-alcohol for 10-30 seconds/rinse with warm H2O/Counterstain with methylene blue for 20-30 seconds/rinse with warm H2O/Blot dry carefully and examine under oil immersion lens
spiral forms--?
Laboratory safety rules – see handout
1-17 ?
18. Wash hands both before and after working in the microbiology laboratory.
19. Gloves are required when working with live organisms.
20. Disinfect your work area before and after working in the laboratory.
21. Do not attempt to transfer cultures while standing.
22. Practice aseptic technique.
23. Remain in your seat while working except when gathering or returning work materials.
24. Bacterial transfers are done by one person holding two tubes.
25. Properly label tubes/plates to be incubated and/or refrigerated with team name, date.
26. Remove all labels from tubes before placing them in the processing area.
27. You must inform your instructor or laboratory paraprofessional if you have any type of lab accident. If you become personally contaminated, your clothing, your books, the floor or work area-the staff needs to know. We will then take the proper precautions to decontaminate you and/or collaterally damaged items or areas.
28. You must inform your instructor if you have any special health conditions or concerns that might affect your laboratory performance in a lab where live microbes are routinely used in experiments. If you are not sure about whether or not this applies to you, discuss it with your instructor to be sure.
29. Use lens paper to remove all traces of immersion oil from microscope lenses.

Special Notes: You must be present at the beginning of each laboratory period to receive the instructions and safety information specific for each session. 90% Laboratory attendance is required to pass this class. Laboratory make-up labs are not available.
Safety Equipment:
Safety glasses/goggles and lab coats are available for purchase from the Southfield OCC Bookstore as well as local retailers.
ELISA – understand the principles and technique—Used for tracking disease outbreaks through testing for antigens and tracking to the origin of the original infectious disease outbreak.
What is the purpose of the washing steps? Ensures that any unbound antibodies are washed away and do not give a false positive test result
Why is a secondary Ab used? So that they will mount an immune response to change the enzyme substrate and color it blue to indicate positive test result where they bind to the primary antibody
?What enzyme system did we use? ELISA/ HRP (Horseradish peroxidase) with a TMB substrate?
What color indicated positive test? Blue

What is Alfa, beta and gamma hemolysis? How was this demonstrated in lab?
Alfa Hemolysis – Greenish area around the colony, indicating incomplete destruction of the RBC’s
Beta hemolysis – clear zone around the colony indicating complete destruction of RBC’s
Gamma hemolysis – no effect on the RBC’s.
What is a bacticinerator? a gasless, flameless sterilizer for use with inoculating loops and needles. It uses infrared heat to accomplish the sterilization without spattering microorganisms. The base is weighted for stability and it houses six loops on its side for convenience.
What is the usual time/temperature achieved in an autoclave for sterilizing lab glassware? (use C for temp)? --- (121C at 15lbs/inch for 15 minutes)
What is room temperature (in C)? About 25C
What are the procedure and reagents for each of the following tests, also indicate what enzyme or product was being detected, know what positive and negative tests results are:
Catalase test (hydrogen peroxide-look for bubbles)
Procedure: Inoculate a TSA plate divided in half equally with your two different cultures and incubate for 48 hrs. at 37degrees. Pour H2O2 over growth on plate to watch for bubbles
Reagents: TSA and Hydrogen Peroxide (H2O2)
Enzyme/product tested for: Catalase Enzyme
Positive indicated by: bubbles formed when the peroxide is broken down into water and oxygen gas
Negative result indicated by: no bubbles formed or only a few scattered bubbles
Oxidase test (dry slide oxidase test – purple color within 20 seconds)
Procedure: Add a sample of your bacteria to the dry slide using a plastic loop. It will develop in 20 seconds.
Reagents: Cytochrome oxidase and artificial electron donors
Enzyme/product tested for: Oxidase
Positive indicated by: dark purple color formation at 20 seconds after sample is added to dry slide
Negative result indicated by: absence of color

Gelatin hydrolysis (use gelatin media, gelatin cannot revert to solid state)
Procedure: Inoculate gelatin deep (tube method)
Reagents: Nutrient gelatin tube/incubate for 7 days (minimum 48 hrs.) / Place in ice bath for entire lab period and observe at end of lab to record which tubes solidified or stayed liquid
Enzyme/product tested for: Exoenzyme; gelatinase—can be used to detect proteolytic enzymes
Positive indicated by: inability of gelatin agar to return to solid state—even below 35 degrees
Negative result indicated by: return to solid state

IMVic test (Indole, methyl red, VP test – used to differentiate enterobacteria based on acid production and fermentation products---see lab handouts)

Methyl Red TEST
Procedure: Add 3-5 drops of methyl red to a tube of prepared media and read immediately
Reagents: Methyl Red
Enzyme/product tested for: (ACID /base) products after test organism degrades glucose
Positive indicated by: Cherry Red for MR
Negative result indicated by: Yellow for MR

VP TEST
Procedure: reagents are added to a broth in which acetyl methyl carbinol is present
Reagents: Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide)
Enzyme/product tested for: what end products result when the test organism degrades glucose
Positive indicated by: turn a pink-burgundy color-- a positive VP test (This color may take 20 to 30 minutes to develop (may take up to 60 minutes)
Negative result indicated by: copper color or yellow is negative (This color may take 20 to 30 minutes to develop (may take up to 60 minutes)
Indole Test
Procedure: The test organism is inoculated into tryptone broth, a rich source of the amino acid tryptophan/ Add about .5ml of Kovac’s, gently agitate and examine the upper layer of liquid for a red color (color should develop within a few seconds)
Reagents: Kovac's reagent (p-dimethylaminobenzaldehyde [an aldehyde] with alcohol)
Enzyme/product tested for: Indole/ tryptophanase, an enzyme that cleaves tryptophan, producing indole and other products
Positive indicated by: Red color = positive
Negative result indicated by: yellow = negative

Carbohydrate fermentation/Sugar Fermentation- same thing
Procedure: : incubate inoculated tubes of broth for 24-48 hours/ Incubate at 37º and observe at 24, 48, 72 hours and at 7 days incubation.
Reagents: Carbohydrate media with acid indicator
Enzyme/product tested for: This is a test commonly used when trying to identify Gram-negative enteric bacteria by production of acid/gas
Positive indicated by: Yellow- acid
Negative result indicated by: Pink-orange -- no acid
Urea hydrolysis
Procedure: incubate inoculated tubes for 24 hours (use three loopfuls to inoculate). Add Phenol Red indicator and read immediately
Reagents: Urea broth
Enzyme/product tested for: Urease- an exoenzyme toxic to most living organisms
Positive indicated by: phenol red indicator turns red in color
Negative result indicated by: orange is negative

Micro Lab Manual in case anyone is missing theirs: http://www.filedropper.com/bio2710labmanual