Answer the following questions as you work your way through the lab material typing in your answers. Then submit your finished lab report as a Microsoft Word document. This lab report is worth 100 points towards your final lab grade. Each Q is worth 2 points unless otherwise noted. Also, per the Honor Code, this work must be your own. This is due Mon. 10/8 at 11:59 PM. The theme of this lab is the identification of unknown bacteria and viruses in a lab.
Selective vs. Differential Media
Selective vs. Differential Media
Use the following website to help you answer Q 1 and 2
1. What is a selective medium? What makes the medium selective? Name 2 examples (3 pts.) A selective medium is a medium that contains antimicrobials, dyes or alcohol that supports the growth of certain organisms, while inhibiting the growth of others. Two examples of selective medium are Mannitol Salt agar and Phenylethyl Alcohol.
2. What is a differential medium? What makes the medium differential? Name 2 examples (3 pts.) Differential medium is distinguishing microorganisms from one another based on growth characteristics. A medium is differential when you are able to visibly see the differences in growth patterns of organisms. Differential media include blood agar and Eosin Methylene Blue.
Steps Used in Identifying an Unknown Bacterium in a Laboratory
I. In a lab situation you would take you inoculum and perform a streak plate in order to separate out individual cells enough to obtain a pure culture
(see Atlas p. 5) 3. What is the most common streaking method? (2 pts.) The most common streaking method is the Streak plate method, while the most common streaking technique is the quadrant method. The quadrant method incubates an agar using a four-streak pattern.
4. What is the principle behind the Streak Plate Method of Isolation? (2 pts.) The Streak Plate Method of Isolation is used to obtain a pure culture in order to isolate a certain organism. This method allows for the organism to produce individual colonies on an agar plate.
II. After incubating your streak plate you would perform a Gram Stain as you learned in Lab #1 – The Virtual Gram Stain. I’m directing you to the Virtual Gram Stain website from the Univ. of Michigan. Click on “View Example. You will need to move your cursor over the test tubes to see what each contains. Then click on the test tubes in the correct order to run the program – this is really cool! http://vudat.msu.edu/gram_stain/
5. What were the results of your Virtual Gram Stain, i.e. describe what you see on the slide as to color, Gram Stain result and morphology? (4 pts.) The gram stain was negative. The shape was bacilli and had purple spores present.
III. Using the dichotomous keys provided as MSWord Documents in this lab, you would carry out specific tests utilizing selective and differential media in order to identity your bacterium. In a microbiology lab you would use “Bergey’s Manual of Determinative Bacteriology”. This book includes all tests and their results to aid in the identification of unknown bacteria. Use the following websites to answer these questions: http://www.saskschools.ca/curr_content/biology20/unit3/unit3_mod1_les2.htm http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf
6. What is a dichotomous key? (4 pts.)
A dichotomous key is a key used to help identify bacteria using process of elimination testing in order to identify each bacteria characteristic.
7. Based on the Information Flow Charts from Bergey’s Manual of Determinative Bacteriology Page 2, answer these questions: (4 pts) a. What is the FIRST test that is performed in a lab to differentiate between groups of bacteria? The first test preformed to differentiate between bacteria is Gram stain testing.
b. If you looked at your slide with the 100X oil immersion objective, what is the next thing that you would observe based on the information in the flow chart? The next step in identification is morphology.
8. The remaining flow chart pages will show you how dichotomous keys are used in bacterial identification. Scroll down to page 7 and look at the Family Enterobacteriaceae which is comprised of Gram negative rods. (4 pts.) a. What is the first test that may be performed to start differentiating among the individual species? The first test that may be performed to start differentiating among the individual species is Lactose Fermentation.
b. Use of a dichotomous key allows you to perform the next test needed to identify your mystery microbe based on the results of the test you just performed, i.e. were the results positive + or negative -. Using the flowchart, what would be the microbe which has these test results: Lactose positive (+), Indole positive (+) and Citrate negative (-)? The microbe would be Escherichia Coli.
ATLAS SECTION 2: SELECTIVE MEDIA.
I would like for you to read over the different types of selective media and then answer the following questions. Remember that selective media promote the growth of some bacteria while actively discouraging the growth of others.
9. For what is Chocolate II agar used? ( 2 pts.)
Chocolate II agar is used for isolation and cultivation of Neisseria and Haemophilus.
10. Based on the information in the Principle section, Phenylethyl Alcohol
Agar will select for the growth of which bacteria? What does the alcohol actually do? (2 pts.) Phenylethyl Alcohol Agar selects for the growth of Gram-positive organisms. The Alcohol in the agar interferes with the DNA synthesis of Gram-negative organisms which inhibits growth.
ATLAS SECTION 7: DIFFERENTIAL MEDIA
Please read over this section. Differential media usually distinguish or differentiate different species of bacteria based on the color of the individual colonies or the areas surrounding them. Look up these tests and answer the following questions: Blood Agar, Catalase, Citrate, Coagulase, Indole, Methyl Red, Motility, TSI, Urea, 11. What is a hemolysis and what type of bacteria produce it? (2 pts.) Hemolysis is the exotoxin of gram positive cocci (streptococcus, enterocus, and aerocccus) that destroy RBCs and hemoglobin.
12. What are the 3 major types of hemolysis and their descriptions? (2 pts.) The three types of hemolysis are B, A, Y. B is complete clearing or destruction of the RBCs or hemoglobin and it results in a clearing of the medium around the colonies. A is partial destruction and a green color forms around the colonies. Y is non-Hemolysis and shows simple growth and no change to the medium.
13. When would you use the Catalase test? (2 pts.)
This test should be used when trying to identify organisms that produce catalase. It is used when differentiating between Catalase positive micrococcaceae and catalase negative streptococcaceae and some variations of the catalase test are for mycobacterium.
14. The Citrate Tests is part of what test series? What is the color of a positive Citrate Test? (2 pts.) The citrate tests are part of the IMVIC (Indole, Methyl Red, Voges-Prokauer and Citrates tests) and are used to distinguish between enterobacteriaceae and other gram negative rods. A positive Citrate test will turn blue.
15. What is the purpose of the Coagulase Test? Why is it to S. aureus’ advantage to produce this enzyme? (2 pts.) The coagulase test is used to differentiate between staphylococcus aureus and other gram positive cocci. Coagulase forms a shield with fibrin barriers to resist phagocytoses and other cellular attacks.
16. The Indole test will help differentiate what group of bacteria? Using the Methyl Red test, what color indicates a positive result? (2 pts.) The Indole test help differentiate enterobacteriaceae and a positive Methyl Red test result is red.
17. What is the principal behind the TSI agar test? The shallow slant and deep butt allow for what? (2 pts.) Triple Sugar Iron Agar test or TSI is loaded with nutrients to help distinguish between enterobacteriaceae and other gram negative rods on the basis of glucose fermentation, lactose fermentation, sucrose fermentation and sulfur reduction. A slanted test tube with a deep butt is used. The agar contains beef and yeast extracts as well as peptone for carbon and nitrogen sources. Also, Sodium thiosulfate for reducible sulfur. Phenol red as a Ph indicator and iron in ferrous sulfate as a hydrogen sulfate indicator. The basis is as something is digested the changes in ph and hydrogen sulfate will cause the color to change.
18. What pathogens can be identified using the Urease test? What color is a positive result?
Identified pathogens come from the genus Proteus. These hydrolyze urea with and enzyme called urease. A positive result will be pink.
Watch this program that will walk you through the process of identifying a foodborne pathogen that has made people sick. Follow the instructions, clicking where indicated to start the activity. Once the file opens, click first on “Gram Stain” and you will see how it works. Then answer the following questions. http://www.swtafe.vic.edu.au/toolbox/lab_ops/laboratory/studynotes/snFlowChartIdentProcBac.htm 19. What Gram positive cocci were discovered using the Gram Stain? (2 pts.) The Gram positive cocci discovered using the Gram Stain are staphylococcus, Micrococcus, Streptococcus.
20. The positive results of the Catalase Test indicated the possible presence of which Gram + bacteria? (2 pts.)
The Catalast test indicated the possible presence of micrococcus and Staphylococcus bacteria.
21. The Oxidation/Fermentation test was positive for which Gram + bacteria? (2 pts.) The Oxidation/Fermentaion test was positive for staphylococcus bacteria.
22. The Coagulase test specifically identified which species of Staphylococcus? (2 pts.) The Coagulase test specifically identified the species aureus.
Now using the Dichotomous Keys provided in the Blackboard section for Lab #3, identify these bacteria based on their test results. Then provide a brief description of each from the Atlas Section 12. (4 pts. each)
23. Test Results: Gram + coccus, Catalase negative, Alpha Hemolysis Bacteria are S. pneumoniae. S. pneumoniae ia an ac-hemolytic, nonmotile, encapsulated, facultatively anaerobic, Gram-positive coccus. It is a significant cause of community-acquired bacterial pneumonia and menigitis in adults. There are at least 80 different serotypes and are defined by antigenically by their capsules. Typically starts in the nasopharynx and from there spread to the lungs and develops into pneumonia or is harbored asymptomatic for months.
24. Test Results: Gram + coccus, Catalase positive, Coagulase negative, Beta Hemolysis Bacteria are S. epidermis. S. epidermis are non-motile, facultatively anaerobic, non-hemolytic, gram-positive coccus. Normal inhabitant of human skin that has become a significant nosocomial pathogen, Most strains produce a slime layer that may enable them to attach to certain hospital apparatus used in surgical procedures, thereby gaining entrance into the body. Most infections at the site of prosthetic implantation are from S. epidermis, can be severe or fatal.
25. Test Results: Gram – rod, Lactose negative, Urease positive Bacteria are P. mirabilis. P. mirabilis are straight, facultatively anaeroic, highly motile (swarming), Gram-negative rod. It is a normal inhabitant of our intestinal tract and is in some other animals as well. It is also common in soil and contaminated water. Is has the “swarming motility” characteristics ad produces a series of visible concentric rings. Common nosocomial pathogen isolated from septic wounds and Urinary tract infections. You get it from direct contact with the source. It can lead to other complications like kidney stones and Proteus Septicemia.
26. Test Results: Gram – rod, Lactose positive, IMViC –++ (= negative, negative, positive, positive), Urease positive Bacteria are K. pneumoniae. K. pneumoniae are nonmotile, encapsulted, facultatively anaerobic, Gram-negative rod. It is found in soil, water, grain, fruits, vegetables and intestinal tracts of a variety of animals including humans. It is in the nasopharynx and oropharynx in humans and is often transmitted as aerosol droplets from person to person. It is a very common nosocominal pathogen. Common infections caused by K. pneumoniae are pneumonia, urinary tract infection, bronchitis, surgical wound infections, biliam tract infections and hospital associated bacteremia. The bacteria are becoming more antibiotic resistant and harder to treat in recent years.
ATLAS SECTION 9: MOLECULAR TECHNIQUES
Today a pathogenic microbe can be identified very quickly using molecular techniques such as DNA Extraction, Electrophoresis, Polymerase Chain Reaction and DNA Sequencing. Answer the following questions using the information in this section of your Atlas.
27. What are the 3 BASIC steps in DNA extraction? (2 pts.)
The 3 basic steps in DNA extraction are-
1. Detergent (Sodium Dodecylsulfate-SDS) is used to lyse cells and
release cellular contents. 2. Heating-dentures proteins and other cell components
3. Water-soluble DNA is precipitated in cold alcohol as a whitish, stringy mass.
28. What does electrophoresis do? What is added to the gel to make the results visible? (2 pts.) Electrophoresis is a technique where molecules are separated by size and electrical charge in a gel. Coomassie blue is added for protein staining and ethidium bromide (fluorescent dye) is used for nucleic acids.
29. What enzyme is used in PCR and why? (2 pts.)
The enzyme used in PCR is DNA Polymerase. It is used because it is able to attach the free nucleotides to complementary bases on the template and create a good copy.
THE VIRTUAL BACTERIA ID LAB
from the HOWARD HUGHES MEDICAL INSTITUTE
Open this website and click on “The Bacterial Identification Lab”. Following the instructions, work your way through this lab.
30. Following the instructions, identify your bacterium and write the species name here. (To do so, you will need to read the page entitled “Nucleotide Sequence (1410 letter)” and click on “Descriptions”. Then click on the top “Accession Code”. Move down to the 7th line: Organism.) (8 pts.) Our bacterium species name is Bartonella henselae
31. Now, on the CDC website (http://www.cdc.gov/healthypets/diseases/catscratch.htm) , look up the information on this bacterium and write a 2 paragraph (4-5 sentences per paragraph) answer on the disease that it causes. (8 pts.)
The disease that our bacterium Bartonella henselae causes is cat scratch disease (CSD). Most people that are affected in CSD have been scratched or bitten by a cat that is carrying it. They develop an infection at the site of injury. The lymph nodes, typically the ones around the neck, head and upper limbs become swollen. Some of the symptoms people with CSD have are fever, headache, fatigue and poor appetite. There are also some rare complications of this bacterium like bacillary angiomatosis and Parinaud’s oculolandular syndrome.
Cats and kittens can spread CDS bacterium to people through bites and scratches. About 40% of cats are carrying CDS at some point in their lives. Cats with CDS do not display and signs of the illness and you cannot determine which cats have it and which do not. You can reduce your risk of contracting CDS by avoiding rough play with cats, washing cat bites and scratches immediately with soap and water, not letting the cat lock open wounds and controlling fleas. You want to call the doctor if an infection occurs after a bite or scratch. Generally CSD is not serious. Medical treatment is not usually needed. Sometimes treatment with antibiotics like azithromycin is helpful in clearing the infection.
Prognosis is good.
On a side note, I actually had this as a child. I got it from a stray kitten scratch. I developed large swollen lymph nodes under my arms, fever and soreness. I am not sure if not as much was known back then but my Dr. did surgery to remove the lymph node from under my left arm and drained the other. It was the only surgery (excluding my c-ections) that I have ever had. I never had any further complications after the surgery and was fine immediately afterwards.
ATLAS SECTION 10: VIRUSES
Viruses cannot be grown on media as bacteria can because they are obligate intracellular parasites and need host cells for reproduction. Therefore their identification in a lab is much more difficult. Often immunological tests are used and you will learn about these in a future lab.
32. Describe the HIV virus. What specific human cells does it infect? (3 pts) The HIV virus is the cause of AIDS. Two forms of the virus exist, HIV-1 and HIV-2. Both are retroviruses and have the ability to make DNA from and RNA template. HIV infects cells with CD4 membrane receptors, normally used for antigen recognition, but by HIV for attachment. A subpopulation of T cells, the T4 helper cells are most commonly affected and die. Other types of cells infected can be dendritic, macrophages and moncytes, HIV can be transmitted through bodily fluids to include: blood, breast milk, semen and vaginal secretions.
33. What is the principle behind growing viral host cells in a lab? What happens after the virus is introduced to the cell culture and what is the result? (5 pts.) The principle behind growing viral host cells is to attain presumptive identification of a virus, how host cells replicate, how quickly it causes damage, and the type of damage it produces. The virus inflicts damage upon the host cell, which in called the CPE (cytopathic effect). It can take as long as 4 days or up to 4 weeks to start seeing damage. Most often they start as small spots (foci) in the cell layer and spread outwards. Common damage to cells includes rounding (small or large), change in texture, or formation of syncytium (the fusion of infected cells).
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