Microscopy Directions

Transcription

1 Name: Exercise 1 Microscopy Focus each slide of bacteria under the microscope using oil immersion. Draw the arrangement of the bacterial cells in the larger portion of the circle and draw the shape of a single bacterium in the smaller portion. When finished, carefully clean each lens of the microscope using a Kimwipe. Bacillus megaterium Streptococcus pyogenes Tot. Mag. Tot. Mag. Staphylococcus aureus Escherichia coli Tot. Mag. Tot. Mag. Summary for Exercise 1:

2 Name: Section: Exercise 4 Exercise 4: Microbes are Everywhere Swab an inanimate object at home and streak on TSA plate. Fomite Sampled Number of Colonies Summary for Exercise 4:

3 Name: Section: Exercise 5 Aseptic Transfer Techniques Observe inoculated tubes for turbidity. Compare results of sterile medium transfers to Staphylococcus aureus inoculation. Sterile Medium Transfers Medium Used nutrient broth A nutrient broth B nutrient agar slant B Observed Growth (+/-) S. aureus Inoculation Medium Used nutrient broth nutrient agar slant nutrient agar deep Observed Growth (+/-) Summary for Exercise 5:

4 Name: Exercise 6 Smear Preparation and the Simple Stain Prepare two smears using organisms from your plate from home Complete the simple stain and observe using the microscope Draw the organisms below Simple Stain Description of shape and color: Simple Stain Description of shape and color: Summary for Exercise 6: Summary for Exercise 11:

5 Name: Exercise 8 The Gram Stain Prepare a smear by mixing S. aureus and E.coli and perform the gram stain Observe under microscope and record results below Description of shapes and colors: Summary for Exercise 8:

6 Name: Exercise 9 The Acid-Fast Stain Observe demonstration slide of Mycobacterium tuberculosis Note the color of an acid-fast organism Description of shapes and colors: Summary for Exercise 9:

7 Name: Exercise 10 The Endospore Stain Observe demonstration slide. Draw observations labeling the cell and endospore. Summary for Exercise 10: Organism name

8 Name: Exercise 12 Bacterial Motility Studies Observe and draw the demo slide. Draw other three types and label the flagella arrangements (monotrichous, amphitrichous, peritrichous, lophotrichous). Summary for Exercise 12:

9 Name: Exercise 13 Morphological Unknown Characterization Prepare a gram stain of one of the five unknown bacteria. Determine which bacteria you stained. G+ or G- Shape Arrangement Bacteria Summary for Exercise 13:

10 Name: Section: Exercise 14 Pure Culture Isolation Skills Streak plate using the quadrant method. Observe demonstration and record growth as (+) or (-). Determine each medium as non-selective, selective for Gram positive, or selective for Gram negative. Organism B. subtilis (gram +) S. aureus (gram +) E. coli (gram -) Selectivity Nutrient Agar MacConkey Agar Phenylethanol Agar Summary for Exercise 14:

11 Name: Exercise 20 Temperature and Bacterial Growth Observe the demonstration of bacteria exposed to different temperatures. Mark the chart if there was growth (+/-) and star (*) if there was pigment production. Categorize the organism as a pyschrophile, mesophile, or thermophile based on temperature growth. Bacterial Culture 5 C Temperature Categorization Pseudomonas fluorescens Escherichia coli Staphylococcus aureus Bacillus stereothermophilus Serratia marcescens Summary for Exercise 20:

12 Name: Exercise 23 Osmotic Pressure and Bacterial Growth Observe plates of E.coli and S. aureus. Determine Halobacterium based on the powerpoint slides. Mark the chart if there was growth (+/-). Categorize the bacteria as a non-halophile, halo/osmotolerant, or extreme halophile. Organism Escherichia coli 0.5% 5% 10% 25% Osmotic Category Staphylococcus aureus Halobacterium salinarium Summary for Exercise 23:

13 Name: Exercise 24 ph and Microbial Growth Observe the demonstration of bacteria grown in various ph levels. Mark if there was growth (+/-). Classify each organism as a neutrophile, acidophile, or alkalophile. Organism ph 3 ph 5 ph 7 ph 9 Classification Staphylococcus aureus Alcaligenes faecalis Escherichia coli Saccharomyces cerevisiae Summary for Exercise 24:

14 Name: Exercise 25 The Importance of Handwashing Draw the colonies on your handwashing plate. Describe your handwashing process and count the number and types of colonies on your plate. Before After # of Colonies # of Types BEFORE AFTER Handwashing process: Summary for Exercise 25:

15 Name: Exercise 31 Microbiology of Milk Count the number of colonies on a good plate and on a poor plate. Multiply by the dilution factor. Determine if the number of colonies complies with the U.S. government milk standards. Suggest what bacteria there might be in the milk. # of Colonies (30-300) Dilution Bacteria per ml Meets Standard? (Y/N) Possible Bacteria GOOD MILK POOR MILK Summary for Exercise 31:

16 Name: Exercise 34 Fermentation of Carbohydrates Include the following in your summary: o The definitions of glycolysis and fermentation o The difference between homolactic and heterolactic fermentation o What the Voges-Proskauer test and Phenol Red broth each measure Summary for Exercise 34:

17 Name: Exercise 40 Chemical Control of Microorganisms Streak plate with culture, divide into four quadrants, place chemical covered filter paper in each quadrant. Measure zones of inhibition and record size for all four types. Note in summary any relation between Gram reaction and susceptibility to chemicals. Organism Gram Reaction S. aureus + E. coli - E. faecalis + P. aeruginosa - 70% Ethanol Chemical Agent Zephiran/ Povidone benzalkonium Iodine chloride Cepacol cetylpyridium chloride Summary for Exercise 40:

18 Name: Exercise 41 Antibiotic Sensitivity Testing Measure and record zones of inhibition of antibiotic disks (diameter in mm). Classify and record bacterial responses to each antibiotic as R (resistant), I (intermediate), or S (sensitive). Refer to chart on page 236. Include in summary the most susceptible bacteria among Staphylococcus aureus, Escherichia coli, P. aeruginosa, Enterococcus faecalis, and Klebsiella pneumoniae. G+ Antibiotics Bacteria: Ampicillin (AM10) Penicillin (P10) Oxacillin (OX1) Cephalothin (CF30) Clindamycin (CC2) Carbenicillin (CB100) Triple Sulfa (SSS1.0) Gentamycin (GM10) Tetracycline (TE30) Erythromycin (E15) Chloramphenicol (C30) Streptomycin (S10) G- Antibiotics Bacteria: Ampicillin (AM10) Gentamycin (GM10) Chloramphenicol (C30) Kanamycin (K30) Nitrofurantoin(F/M300) Cephalothin (CF30) Streptomycin (S10) Tetracycline (TE30) Triple Sulfa (SSS1.0) Carbenicillin (CB100) Colistin (CL10) Nalidixic (NA30) Summary for Exercise 41:

19 Name: Exercise 45 UV Light Lethality and Photoreactivation Expose plates to treatments, wrap in tinfoil, and incubate until next class. Count the number of colonies on each plate and record results. Summary for Exercise 45: Organism: Treatment UV UV + PHR 0 min Plate # 1 Plate #5 1 min Plate # 2 Plate #6 2 min Plate #3 Plate #7 2 min + cover Plate #4

20 Name: Exercise 48 Staphylococci on Skin Wet swab, sample the skin, and streak TSA and MSA. Using a new swab, repeat for nares. Record growth- number, size, and color. Yellow MSA medium indicates S. aureus. Medium Skin Growth Observations Nares Growth Observations TSA MSA Summary for Exercise 48:

21 Name: Exercise 49 Upper Respiratory Tract Culture Record the demonstration hemolytic reaction (α, β, γ). Compare to the results of your throat culture and determine the hemolytic reactions. Bacteria Streptococcus pneumoniae Streptococcus pyogenes Streptococcus mitis Staphylococcus aureus Summary for Exercise 49: Hemolytic Reaction Throat Culture Growth (# of colonies and color) Hemolytic Reactions Observed

22 Name: Exercise 52 Snyder Test for Dental Caries Susceptibility Observe demonstration of Snyder Test tubes, note colors. Indicate (X) in chart when the agar changes color at each level of susceptibility. Summary for Exercise 41: Slight Moderate Marked Susceptibility to Dental Caries 24 hours 48 hours 72 hours

23 Board Process ( to write on the board.) LAB 1 Exercise 1 Microscopy 1. Observe bacteria under the microscope using the oil immersion lens (100x). 2. Draw shape and arrangement of organism in your lab report and specify the total magnification. 3. Repeat for other three organisms. *Remember to wipe slide and lens clean with a KimWipe. Staphylococcus aureus #4 Streptococcus pyogenes #3 Shape Bacillus megaterium #9 Escherichia coli #5 Arrangement [Numbers correspond with the colorful BACTERIA chart] Exercise 4 Microbes are Everywhere At Home: 1. Wet sterile swab with tap water 2. Rub swab on an inanimate object. 3. Streak swab on nutrient agar (TSA). 4. Incubate upside down (lid on bottom) at room temperature for one week.

24 LAB 2 Exercise 4 Microbes are Everywhere 1. Record the fomite you sampled and your observations. 2. Count and record the number and types of colonies on the plate from home. Exercise 5 Aseptic Transfer Broth B Broth C Broth A Deep Slant B Slant C S. aureus 1. Flame loop and tube openings in between each transfer. Use needle to stab the deep. 2. Perform transfers as shown above. Transfer Broth A to Broth B and Slant B. Transfer S. aureus to Broth C, Slant C, and Deep. Exercise 14 Pure Culture Isolation Skills 1. Observe demo and record growth. 2. Inoculate plate with mixed culture using the streak plate method. Remember to flame loop between each quadrant Growth on Selective and Non-Selective Media Organism Nutrient Agar MacConkey Agar Phenylethanol Agar B. subtilis (gram +) S. aureus (gram +) E. coli (gram -) + + -

25 Lab 3 Exercise 6 Smear Preparation and the Simple Stain 1. Sterilize inoculating loop and place small drop of water on slide. 2. Sterilize loop and mix a small loop of bacteria in water and spread over 2/3 of slide. 3. Let air dry then heat fix slide. 4. Flood the slide with crystal violet for one minute. Then gently rinse with water until it runs clear. 5. Blot slide dry with a KimWipe and observe using oil immersion lens (100X) under microscope. 6. Repeat smear preparation and perform stain with safranin. Exercise 11 The Capsule Stain 1. Observe demonstration slide or #8 on BACTERIA chart of Klebsiella pneumonia. 2. Label capsule, cell and background in lab report.

26 Lab 4 Exercise 8 The Gram Stain 1. Prepare a smear mixing both E. coli and S. aureus on slide. Remember to heat fix. 2. Flood slide with primary stain, crystal violet, for one minute. Gently rinse with water. 3. Flood slide with mordant, iodine, for one minute. Rinse with water. 4. Decolorize slide using acetone, rinsing continuously for no more than 15 seconds. Rinse with water. 5. Flood slide with counterstain, safranin, for one minute. Rinse with water. 6. Blot dry with KimWipe and observe under microscope using oil immersion lens (100x). -E. coli is a Gram negative rod. -S. aureus is a Gram positive coccus. Exercise 9 The Acid-Fast Stain 1. Observe demonstration slide or #25 on Microbiological Chart of Mycobacterium tuberculosis. 2. Specify colors of non-acid-fast versus an acid-fast organism. Exercise 10 The Endospore Stain 1. Observe demonstration slide or #32 on Microbiological Chart of Clostridium tetani. 2. Label endospore and cell portions.

27 Lab 5 Exercise 12 Bacterial Motility Studies 1. With a partner, inoculate motility deeps with a single bacterium in each tube, using Pseudomonas aeruginosa Proteus vulgaris Micrococcus luteus 2. Flame loop in between inoculations. 3. Incubate in cans at room temperature. 4. Observe demo slide of a flagella stain. Determine the flagellar arrangement. Exercise 13 Morphological Unknown 1. Choose one of the five unknown cultures and prepare a smear. 2. Perform the Gram stain. a) crystal violet (1 min) b) iodine (1 min) c) acetone (15 sec) d) safranin (1 min) 3. Observe slide under microscope using the oil immersion lens. Determine bacteria type based on Gram reaction, morphology, and cell arrangement. Gram positive Bacillus subtilis (rods in chains) Staph. aureus (cocci in clusters) Micrococcus luteus (cocci in tetrads and clusters) Gram negative E. coli (short rods) Klebsiella pneumoniae (rods)

28 Lab 6 Exercise 12 Motility Studies 1. Observe motility deeps for growth. 2. Classify each bacteria as non-motile or motile (aerobic or facultative). Exercise 20 Temperature and Bacterial Growth 1. Observe demonstration of bacteria incubated at 5, 25, 37, and 55 C. 2. Record results in chart and classify each bacteria as psychrophile, mesophile, or thermophile. Note temperature dependent pigment production. Organism 5 C 25 C 37 C 55 C S. aureus B. stereotherm P. fluorescens E. coli S. marcescens - + (pigment) + (no pigment) - Exercise 23 Osmotic Pressure and Bacterial Growth 1. Divide salt plate in half, labeling each side. 2. Streak one side with S. aureus and the other side with E. coli. 3. Put plate in incubator. *Next week you will compare the growth on 0.5%, 5%, 10%, and 25% NaCl. S. A. E. C.

29 Lab 7 Exercise 23 Osmotic Pressure and Bacterial Growth 1. Observe plates of 0.5, 5, 10, and 25% NaCl. 2. Record growth of E. coli and S. aureus. 3. Classify each organism, including Halobacterium salinarium, according to salt tolerance as non-halophile, halo/osmotolerant, or halophile. Organism 0.5% 5% 10% 25% E. coli S. aureus Halobacterium salinarium Exercise 24 ph and Bacterial Growth 1. Observe demonstration tubes of bacteria grown in ph 3, 5, 7, and Classify each organism as acidophile, neutrophile, or alkalophile. Organism ph 3 ph 5 ph 7 ph 9 A. faecalis E. coli S. aureus - +/- + - S. cerevisiae Exercise 25 The Importance of Handwashing 1. Divide plate in half and label. 2. At home, press four fingers onto the BEFORE side. 3. Wash and dry hands as you normally do and then press the same four fingers onto the AFTER side. 4. Incubate at room temperature and bring to lab next week. Before After

30 Lab 8 Exercise 25 Handwashing 1. Count the number and types of colonies before and after handwashing. 2. Describe how you washed/dried your hands and how you could improve. Exercise 34 Fermentation of Carbohydrates 1. At home, review powerpoint slides. 2. Study the Summary Slide and use this to write your conclusion. 3. Complete the lab report questions and conclusion. Exercise 31 Microbiology of Milk GOOD MILK 1 ml 1 ml 0.1 ml 1 ml 0.1 ml undiluted 1:10 1:100 1:1,000 POOR MILK 1 ml 1 ml 1 ml 1 ml 0.1 ml 1 ml 0.1 ml 1:10,000 1:100,000 1:1,000,000 1:10,000, Following diagram, make dilutions and plate proper amounts. Change pipettes according to color. 2. Spread milk using glass beads. When finished, dump glass beads in orange cans. 3. Place plates in incubator and make sure they are labeled with name and dilution.

31 Lab 9 Exercise 31 Microbiology of Milk 1. Choose a plate with 30 to 300 colonies. 2. Multiply the number of colonies by dilution factor. This gives you the bacteria per ml. 3. Does the milk pass the U.S. government standard for pasteurized milk? 55 colonies x 100 (dilution factor) = 5500 bacteria/ml Exercise 40 Chemical Control of Microorganisms 1. Streak plate with one of the four bacteria. 2. Divide plate into four quadrants and label B, CP, 70, I. 3. Dip paper disk into each chemical agent and then place the disk in the labeled quadrant. 4. Place in incubator with disks facing up. CP I 70 B Exercise 45 UV Light Lethality and PHR 1. Evenly streak plate with assigned bacteria. (S. aureus or E. coli) 2. Perform treatment according to plate number. 3. Wrap all plates in aluminum foil after finishing treatment and then place in incubator. Plate # Treatment 1 UV, 0 min 2 UV, 1 min 3 UV, 2 min 4 UV, 2 min (cover on) 5 UV, 0 min + PHR, 1 min 6 UV, 1 min + PHR, 1 min 7 UV, 2 min + PHR, 1 min

32 Lab 10 Exercise 40 Chemical Control of Microorganisms 1. Measure zones of inhibition for each chemical on each plate (diameter in mm). 2. Which disinfectant was most effective for G- bacteria? 3. Which was most effective for G+ bacteria? S. aureus G+ E. faecalis G+ E. coli G- P. aeruginosa G- Exercise 45 UV Light Lethality and PHR 1. Count the number of colonies on each plate. 2. Determine the effects of UV light and photoreactivation. Plate # Treatment Sample Results 1 UV, 0 min TMTC 2 UV, 1 min 98 3 UV, 2 min 13 4 UV, 2 min (cover on) TMTC 5 UV, 0 min + PHR, 1 min TMTC 6 UV, 1 min + PHR, 1 min UV, 2 min + PHR, 1 min 44 Exercise 48 Skin/Nares 1. Divide TSA and MSA plates and label. 2. Wet swab in drinking fountain, sample nares and streak on half of each plate. 3. Repeat for skin using a new swab. skin nares Exercise 49 Upper Respiratory Tract 1. Moisten sterile swab in water. 2. Sample tonsils with swab using tongue depressor. Avoid contact with teeth or tongue. 3. Streak sample onto sheep blood agar. *Remember to stab in quadrant 2

33 Lab 11 Exercise 48 Skin/Nares 1. Observe TSA and MSA plates for growth. 2. Which plate grew the greatest variety of bacteria? 3. Do you have any S. aureus on your plate? 4. Observe demonstration plates and record observations. Exercise 49 Upper Respiratory Tract 1. Observe sheep blood agar of the throat culture. 2. Compare to demonstration plates and determine any hemolytic reactions on your plate. β clear halo (full hemolysis of red blood cells) α green colonies (partial lysis) γ creamy colonies (no lysis) Exercise 41 Antibiotic Sensitivity Testing 1. Measure zones of inhibition (diameter in mm) on bacterial plate. 2. Using chart on page 236, determine the response of the bacteria as resistant (R), intermediate (I), or sensitive (S). 3. Compare antibiotic resistance between G- and G+ bacteria. Exercise 52 Snyder Test Observe demonstration tube. Susceptibility to Dental Caries 24 hours 48 hours 72 hours Slight X Moderate X Marked X