23

Lab 8
Exercise 18 Culture Media Preparation Part 2 Background: Culture Media nutrients used to grow cells or microbes A. Agar nutrients in gel-like form Plates, slants, and deeps B. Broth nutrients in liquid form Procedure:
Weigh out then mix media with water in a flask that is at least twice the capacity of the volume of the water that is used. If media is an agar, heat to boiling for 1 minute, stirring constantly, to dissolve. Set hot plate to "10". Broths do not require heating. Boiling media will foam up, so be ready with a hot pad to quickly remove media from hot plate. Dispense required volume into tubes using a pipet. If preparing plates, just cap flask with foil. Put caps on tubes. For tubes, write 4 labels with class, type of media, and date, and then put on top of caps. Use a pencil. For flasks used for plates, write 2 labels with class, type of media, and date, and then put one on foil and one on flask. For "bottle media", use the screw-cap square bottles. Just place the caps on the bottles. Do not screw the caps on. Put the media in the spot designated by the instructor. Instructor will autoclave media. Rinse out pipettes with water using pipette pumps.

Group Media

Vol. Media Water (g) (mL)

Total No. Tubes or Plates to Make

75 48 200 525 375 375 200 75 40 75 60 200 630 693 48 48 15 8 8 7 15 15 8 15 8 15 15 8 7 7 8 8

Vol. / tube (mL / tube)

2 3

Trypticase Soy Broth Fluid thioglycollate Agar Deep Trypticase Soy Agar Plates Trypticase Soy Agar bottles EMB Agar Plates MSA Agar Plates MacConkey Agar Plates Phenol Red Glucose Broths Phenol Red Lactose Broths MR-VP broths Simmons Citrate Agar Slants Mueller-Hinton II Agar Plates 90 mL sterile water bottles 99 mL sterile water bottles Soft Agar Deeps (use "Agar, granulated") Motility Media Agar Deeps

2.3 1.4 8.0 21.0 14.0 41.6 10.0 1.5 0.8 1.3 1.5 7.6 0.0 0.0 0.3 1.1

5 6 25 75 25 25 25 5 5 5 4 25 90 99 6 6

24 Lab Report: none Know for Exam: Know how to weigh, pipette, mix, and dispense media. Do not have to memorize media recipes. Know how to pour plates. Know the different types of culture media preparations.

Dilutions Preparation: Background: Procedure: Take notes on Dilutions Know for exam: Be able to solve dilution problems Be able to calculate concentrations of bacteria in stock samples

Exercise 18 Culture Media Preparation Part 2 Procedure: Each group label plates as indicated below on bottom with a wax pencil & pour. Rinse out empty flasks, remove labels, and put away. Group 1 2 3 4 5 6 Number of plates 8 15 8 7 8 8 Label TSA (trypticase soy agar) EMB (eosin methylene blue agar) MSA (mannitol salt agar) MSA (mannitol salt agar) MH (Mueller-Hinton II agar) Mac (Maconkey agar)

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Lab 9
Exercise ?? Selective and Differential Media Inoculations (not in labbook) Background: Selective Media Selects (promotes growth of) some bacteria, inhibits others Differential Media Differentiates different microbes based on appearance Procedure: A. Each group label the following plates on bottom with wax pencil EMB MSA MacConkey Also include paper label with regular information (section no., etc.) B. Aseptically inoculate EMB, MSA, and Mac plates with 1. E. coli 2. Proteus mirabilis 3. Staphylococcus epidermidis 4. Bacillus subtilis E. Incubate all plates 48 hours at 37C

1 4

2 3

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Summary of Selective and Differential Media

Media MacConkey Agar

Selective Properties Promotes Inhibits Gram negative Gram positive

Differential Properties Appearance Conclusion* Pink colonies Lactose fermenter White or gray colonies Lactose nonfermenter Mannitol fermenter Mannitol nonfermenter Strong lactose fermenter (E. coli) Lactose fermenter

Mannitol Salt Agar

Halotolerant

Non-halotolerant

Yellow medium Original pink medium

EMB Agar

Gram negative

Gram positive

Metallic green colonies Dark purple/black colonies Colonies that are light pink or color of media

Lactose nonfermenter

*Note that there would have to be visible growth in order to determine whether or not bacteria fermented these sugars.

27 Exercise 44 Bacterial Counts of Foods Inoculations Preparation: read page 317 318 and review dilutions notes Procedure: Follow labbook procedure except: # 1. Weigh 10 g or 10 mL of food (instead of 20) in sterile Petri plates. # 2. Instead of putting food in blender, add it to 90 mL sterile water blank and shake 25 times (this is still 1:10 dilution) Using sample in 90 mL water blank (blender), make streak plates on EMB and MSA. Clean water blanks, agar bottles and caps with a brush, soap, and water, and then dry and put away. Food assignments: Group 1 2 3 4 5 6

Food Hamburger Sushi Potted meat Bagged salad Unwashed Fresh vegetable (chopped broccoli florets) Washed Fresh vegetable (chopped broccoli florets)

Exercise 21 Bacteriophage (preparation) Procedure: Each group inoculate one Trypticase Soy Broth with E. coli B

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Lab 10
Exercise ?? Selective and Differential Media Evaluations Procedure: Evaluate media using Summary of Selective and Differential Media. Enter results on Selective and Differential Media Results table. Lab Report: none Know for exam: Define selective and differential media. Know information on Summary of Selective and Differential Media table. Be able to interpret results on the media shown below:

MacConkey Agar

Mannitol Salt Agar

EMB Agar

Photograph by Spence Dowlen

Photograph by Jeni Sharpe

Photograph by Jeni Sharpe

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Selective and Differential Media results

Medium MacConkey Microbe 1. E. coli 2. Proteus mirabilis 3. Staphylococcus epidermidis 4. Bacillus subtilis Mannitol Salt Agar 1. E. coli 2. Proteus mirabilis 3. Staphylococcus epidermidis 4. Bacillus subtilis EMB Agar 1. E. coli 2. Proteus mirabilis 3. Staphylococcus epidermidis 4. Bacillus subtilis --------------------------------------------------------------------Amount (+++, ++, +, -) and color of Growth Color Change in Medium --------------------------------------------------------------------Conclusion

30 Exercise 44 Bacterial Counts of Foods Evaluation Preparation: read page 317 318 and review dilution notes Background: Example of results:

500 Colonies

50 Colonies

5 Colonies

1:100

1:1,000

1:10,000

Choose plate that has between 30 and 300 colonies Count even small colonies Calculate organisms per mL Organisms/ml = # colonies X inverse of dilution Selective and Differential media results MSA Halotolerant, mannitol fermenter often Staphylococcus aureus (food poisoning and Staph. infections) EMB shiny, green colonies E. coli, often from fecal contamination Procedure: Calculate the concentration of bacteria in your food sample. Evaluate the results of the MSA and EMB plates that were inoculated with the food sample. Enter results on Bacterial Counts of Food Results table. Lab Report: do all Know for exam: Be able to determine the bacterial concentration in foods by diluting samples and applying to plates. Understand the dilution scheme of Figure 46.1 in the labbook. Be able to interpret results of food samples applied to MSA and EMB agars. Be able to answer questions about the typical, ideal results for each food tested in the lab (will not have to give exact numbers) and why we would expect high or low bacterial counts in each of these foods.

31

Bacterial Counts of Food Results

Type of Food Plate Count Dilution Bacterial Concentration (CFU/mL) MSA Results* EMB Results**

*MSA Halotolerant, mannitol fermenters are often Staphylococcus aureus. **EMB Shiny, green colonies are usually E. coli which indicate fecal contamination.

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Photograph by Jeni Sharpe

Exercise 21 Determination of a Bacteriophage Titer Inoculations Preparation: read pages 165 167 Background: Procedure: Use Figure 23.2 to see an overview of procedure, but note that we will not make any dilutions and will make only one plate. 1. Label 1 TSA plates with standard 4 items of information. 2. Remove 1 liquid soft agar tube from the hot water bath and transfer 1 mL of the phage culture to the tube using a sterile transfer pipette. 3. Using the same transfer pipette, transfer 2 3 drops of the E. coli B broth culture to the soft agar tube and mix by rolling tube in hands. 4. Pour the contents of the soft agar tube onto the TSA plate. Gently rock the plate so that the soft agar completely covers the surface. 5. Allow the plate to sit for a few minutes so the soft agar can solidify. 6. Since the initial bond between the two agar layers is weak, place the plate right side up in the 37 C incubator. The instructor will turn them upside down in 1 hour.

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Lab 11
Exercise 21 Determination of a Bacteriophage Titer Evaluations Preparation: read pages 165 169 Background: Plaques clear areas of dead bacteria caused by bacteriophage DRAW

Procedure: Examine plates looking for plaques. Lab Report: B(1-3, 5, 6) Know for exam: Understand the procedure for preparing this plate of bacteriophage. Define plaques. Understand lab report material.

Photograph by Jeni Sharpe

34 Exercise 34 Morphological Study of Unknown Bacterium Inoculations Preparation: read pages 239 243 Background: Study of Unknown Bacterium General Plan Receive unknown bacteria (Group 1 gets Unknown # 1, etc.) Run tests on it over next several labs Compile results and identify Procedure: A. Gram stain unknown and observe Record on Test Results on Unknown table: Gram reaction Cell shape Cell arrangement Presence of any endospores B. Motility Inoculate motility media by stabbing with inoculating needle about 2/3 of the depth of the media. Incubate 1 day at room temperature. Know for exam: Know how to perform a Gram Stain on unknown and interpret the following results. o Gram reaction o Cell shape o Cell arrangement o Presence of endospores

Exercise 35 Cultural Characteristics Inoculations

Preparation: read page 245 Procedure: Inoculate a tube of fluid thioglycollate media (FTM) with a loopful of the unknown. Mix organisms by rolling the tube between your palms. Include type of media (FTM) on label. Incubate at room temperature.

35 Exercise 36 Oxidation and Fermentation Tests Inoculations Preparation: read pages 249 258 Background: Oxidation and Fermentation Tests Testing for various fermentation products and enzyme production Test that will be performed: A. Glucose fermentation B. Lactose fermentation C. Mixed acid fermentation (MR test) D. Citrate test E. Catalase test Procedure: Inoculate the following media with the unknown: o Phenol Red Glucose o Phenol Red Lactose o MR-VP o Citrate Inoculate the following media with known bacteria to use as positive test controls: o E. coli Phenol Red Glucose MR-VP o Enterobacter aerogenes Citrate Include name of media on each tube. Each group will need: o 2 Phenol Red Glucose o 1 Phenol Red Lactose o 2 MR-VP o 2 Citrate Some types of media look identical. Label or mark media before take back to table. Put tubes in incubator, consolidating tubes into as few racks as possible.

36 Exercise 31 Antibiotic Sensitivity Testing (preparation) Procedure: Each group inoculate one Trypticase Soy Broth as follows: Group 1 2 3 4 5 6 Incubate at 37 C Bacteria Bacillus subtilis E. coli Proteus mirabilis Enterobacter aerogenes Bacillus subtilis E. coli

37

Test Results on Unknown

Unknown # ________________ Bacterial ID _______________________________ Conclusions

Observations Gram Reaction Shape Arrangement Presence of Endospores Motility Test Oxygen Requirements Glucose Fermentation Lactose Fermentation Mixed Acid Fermentation (MR Test) Citrate Test Catalase Test

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Lab 12
Exercise 34 Morphological Study of Unknown Bacterium Evaluations Preparation: read page 242 Motility Procedure: Evaluate motility media tube for motility and record on Test Results on Unknown table. Know for exam: Know how to determine the motility of an unknown using motility media.

Photograph by Jeni Sharpe

Exercise 35 Cultural Characteristics Evaluations Preparation: read page 247 Background: Using fluid thioglycollate media (FTM) to determine oxygen requirements: Thioglycollate binds available oxygen FTM has an indicator that turns green if oxygen present There is more oxygen at top of media, none at the bottom DRAW

Obligate Aerobe

Microaerophile

Facultative Anaerobe

Obligate Anaerobe

Procedure: Examine thioglycollate tube, determine and record oxygen requirements on Test Results on Unknown table. BE GENTLE WITH TUBES. DO NOT SHAKE OR MIX Know for exam: Know how fluid thioglycollate media works and be able to interpret results Only drawings of growth distributions will be used on the exam.

39 Exercise 36 Oxidation and Fermentation tests Evaluations Preparation: read pages 249 258, and Diagnostic Media Summary Procedure: Do Second Period Test Evaluations for: o Carbohydrates in Durham Tubes o Mixed Acid Fermentation o Citrate Test o Catalase test (use unknown slant and Proteus mirabilis stock cultures.) Use Diagnostic Media Summary Table to help determine results. Record results for unknown on Test Results on Unknown table as positive or negative. Know for exam: Know information on Diagnostic Media Summary and be able to determine the following when given inoculated media: o Test o Media o Substrate o Enzyme o Product o Any added reagents o Whether test is positive or negative On exam, will be told either what media is being used or what test is being run. Phenol Red Glucose Broth

Photographs by Karen Kendall-Fite

Phenol Red Lactose Broth

MR-VP Media

Simmons Citrate Agar

Photographs by Karen Kendall-Fite

Photographs by Karen Kendall-Fite Photograph by Karen Kendall-Fite

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Diagnostic Media Summary

Test Glucose Fermentation Lactose Fermentation Mixed Acid Fermentation Citrate Test Catalase Test Media Phenol Red Glucose broth Phenol Red Lactose broth MR-VP Simmons Citrate Agar slant Nutrient agar slant Reactions Occurring in Positive Tests* (Substrate enzyme Product) glucose fermentation enzymes Glucose acids Lactose Glucose Citrate H2O2
lactose fermentation enzymes

Indication of Positive Test Yellow media Yellow media Red media Blue media Bubbles

acids strong acids + Methyl Red

mixed acid fermentation enzymes

citrase

alkaline products O2 + H2O

catalase

Indication of Negative Test Original red media Original red media Original light yellow media Original green media No bubbles

*Bold compounds are reagents added by experimenter. Be able to recognize positive and negative reactions and to name the substrate, enzymes, products and reagents added in each reaction.

41 Exercise 39 Use of Bergeys Manual (Identification of Unknown Bacterium) Preparation: read pages 275 279 Background: Bergeys Manual Best classification scheme Divide bacteria up into 11 groups Several genera, many species in each group Procedure: Use Figures 41.1 and 41.2 to identify group of unknown Use text descriptions to identify genus Check answer with instructor Know for exam: Given tables and information on pp. 275 279 and lab results in a Test Results on Unknown table, be able to determine genus of unknown bacteria.

Exercise 31 Antibiotic Sensitivity Testing Inoculations Preparation: read pages 215 220 Background: Antibiotic Sensitivity Testing: The Kirby-Bauer Method Measures the sensitivity of bacteria to an antibiotic by measuring how well growth in inhibited (zone of inhibition) DRAW

Procedure: Follow labbook procedure for First Period plate preparation except use broth inoculated in last lab to make lawns. Applicator will lock up if one of the cartridges is empty. Do not force mechanism. (Replace empty cartridge and then continue.) Gently press down discs with sterile loop (WCC and LCC)

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Lab 13
Exercise 31 Antimicrobic Sensitivity Testing Evaluation Preparation: read pages 215 220 Procedure: Follow labbook Second Period Interpretation The letter codes for the antibiotic disks are as follows: Tetracycline (TE) Sulfisoxazole (SXT or G) Streptomycin (S) Gentamicin (GM) Ampicillin (AM) Ciprofloxacin (C) Measure diameter of the zones of inhibition (ZOI) from bottom of plate. If no inhibition, just record diameter of disc (7 mm) DRAW

Use Table 33.1 to determine if bacteria are resistant, intermediate, or susceptible to the antimicrobial. Enter results on table on p. 221. If bacteria is not covered in Table 33.1, leave blank

Know for exam: Be able to use the Kirby-Bauer method to determine whether bacteria are sensitive, intermediate, or resistant to an antibiotic. On the exam, you will be given a plate, ruler, Table 33.1, and the letter code for the antibiotics used.

Photograph by Karen Kendall-Fite

Photograph by Karen Kendall-Fite

43 Lab Cleanup by Students Procedure: Clean bench top Clean bench top gutter Clean out sinks Clean microscopes (each group clean two) Wash out staining trays Wrap cords around Bacticinerators Clean inside and outside of incubator Fill water bottles Fill disinfectant bottles At Columbia and Lawrenceburg for each bin o Remove trash o Wipe out with disinfectant o Make sure each bin has the following: 1 inoculating needle 2 loops 2 clothespins 1 lens paper booklet 2 half-rack 1 wax pencil 10 sheet of labels 1 ruler o Put extra drawer items on front desk At Franklin for each groups drawers o Remove trash including from shelf above chair o Wipe out with disinfectant o Make sure each station has the following: Top Drawer 1 inoculating needle 1 loop 1 clothespin 1 lens paper booklet 1 wax pencil 1 immersion oil 1 stirring rod 1 flask clamp 1 test tube clamp 1 weigh boat 6 sheet labels. 1 ruler

44 Second Drawer 1 pair Hot pad gloves Cabinet Burner Rack Bacticinerator (one each table) Burner stand. 100 mL graduated cylinder

Open Lab Procedure: Review examples of inoculated media. Know all information, procedures, handouts, and lab reports given in the Lab.

45

Lab 14
Lab Exam # 2