SCHEDULE

MICROBIOLOGY LABORATORY

Laboratory plays a very important part in this course. For a successful experience, numerous techniques must be learned and mastered, therefore attendance is mandatory. A laboratory that is missed for any reason cannot be made up and will be recorded as a zero for the exercise(s) covered that day or week. A student who misses two labs will not be allowed to continue in the course. Lab reports are due the day the lab exercise is completed. Any late reports will not be accepted and will be recorded as a zero.

Lab begins promptly on the hour so please be on time. The formal instructions and demonstrations of the day’s work will be given immediately at the beginning of the lab period. Late comers to lab will not be allowed into lab until the lab instructions are completed. At the beginning of lab, all you need is your lab manual and your note taking material and I need your undivided attention. Leave your microscope in the case until you are ready to use it.

PLEASE NOTE: Several of the exercises will require that a student spend extra time in the laboratory on days other the scheduled Tuesday/Thursday times. That means that a student will be required to come to lab on some Wednesdays and/or Fridays or even on a Monday. These exercises are designated with an asterisk next to the date. THE EXTRA LABS TIME WILL BE MANDATORY

During certain labs a calculator would be helpful to expedite the calculation of lab results, however it is not mandatory. The specific need for a calculator is pointed out in the individual exercises.

EXERCISE PAGE

Introductory Laboratory 1

Read pages 95 & 96 very carefully and also 97 – 101. Get assigned a permanent lab seat, storage drawer, loop handle and microscope. Get familiarized with your microscope and learn how to use the oil immersion lens. Microscopic lens cleaning technique p. 98.

Pond Water Exercise 5

Read : Eukaryotic and Prokaryotic Cells on p.6. Get a look into the microbial world in a drop of pond water -- the eukaryotic - procaryotic differential-- what Leeuwenhoek saw. Additionally learn how to degrease a slide for future bacterial smear preparation.

Cellular Morphological Characteristics of Common Bacteria 9 Read: Cell Morph. 106; Smear 103; Simple Stain 104; Test Tube Handling 121; Nutrient Broth 132. Today you will be taught several important techniques: the proper handling of microbial cultures; the proper handling of test tubes and caps; the proper handling, sterilization and use of the inoculating loop; the proper technique for smear preparation. Additionally, today you will be issued your first unknown.

Staining Procedures - The Gram Stain 13

Read: Gram Stain 104-105. The gram stain is the most widely used stain in microbiology - make sure you master it because you will be doing it often. Remember to apply all of the proper techniques that you learned Tuesday so that you carry them out correctly.

Dilution Plate Counting & Distribution of Bacteria 17

Read: 109 - 113; Pipette Pump Instructions 107; Physiological Saline 137

Pipetting technique, dilutions, and dilution factor calculations are some of the things you must learn completely, so pay attention. In the follow-up lab you will be counting the colonies and consequently calculating the total plate counts for each of the samples. All these type things that pertain to dilutions and dilution problems will be on the lab exam. A calculator will be helpful.

Examination of Cultures for Motility 25

Read: Streaking 114, 115, and 116. This exercise is the ultimate use of the microscope. You will need to clean your microscope objective lenses, especially the high dry objective, before beginning.

Plate Streaking 21

Today you learn to streak a plate. This is another critical technique so pay close attention because it will play a significant role in the next three weeks of lab exercises.

Examination of Bacterial Colonies 21

Read : Oblique Light Microscopy 117; Colonial Char. 118-119

Routine Study of Pure Cultures 122-123

THIS WILL BE PRESENTED DURING LECTURE - BRING LAB MANUAL

Read: Pure Culture Techniques 120; Culture Manipul. 121 Culture Media 128-129; Carbo. Ferment. Broth 74; Gelatin 131; Tryptone Broth 134

You will be instructed about the next three weeks of lab and the lab procedures in a lab lecture given during lecture time.

Differentiation and Identification of Pure Cultures of Some Common Bacteria (first 6) 29

The study of six different microorganisms for the preparation of your own “Bergey’s Manual” of characteristics. Work in groups of 4 or by table. Individually do a good job because the three other people of your table group are depending on you. During the follow-up lab we will compiling the results for your report.

Differentiation and Identification of Pure Cultures of Some Common Bacteria (second 6) 29

Continue with the next six organisms. Switch off the work within your group as to last week’s assignments.

We will be beginning our applied study of Fermentation and Respiration 87

We will inoculate some grape juice with yeast to ferment the sugar to alcohol. Consequently, on Oct. 27 we will inoculate the wine with an oxidative organism to convert the wine to vinegar. The whole process will take about two months. We will follow it on a weekly basis until Nov. 22.

“Unknown” Mixture for Isolation and Identification of Component Species 35

This is the most important unknown you will have all semester. It is worth 50% of your total unknown average for the semester. It will be worth your while to put some significant effort into the identification of the organisms in this unknown. The unknown mixture will be available Monday Oct. 10, beginning at 7:30 a.m. and throughout the rest of the day. It will also be available on Tuesday during lab time. If you wait until Tuesday, you will not get any help in “selecting” the colonies so be there on Monday if at all possible. It will take about 10 minutes to complete the initial work. You will also need to come into lab on Wednesday, Thursday and maybe Friday, depending how well things go for you. A schedule of lab availability times during the week will be posted for your information. The report of your findings is due by the end of your scheduled lab on Oct. 18.

Growth of Bacteria 43

Read : Graphic Plotting of Data 124-127; Calculation of Generation Time on page 125-126. This is a multi-lab section effort, with the participation of all the labs. We will begin this growth exercise at exactly 11:00 a.m. taking timed samples every 30 min. up through 5:00 p.m. Do a good job - a number of other students are depending on your results. YOUR COLONY COUNT MUST BE COMPLETED AND RECORDED BEFORE 9:25 A.M. Oct. 20 ON THE APPROPRIATE COMPREHENSIVE RESULT SHEETS AVAILABLE AT YOUR INDIVIDUAL TABLE. All of the collected data for 30 and 37 C will be plotted graphically by each student. Semi-log graph paper is available in back of the manual. Bring a calculator to the follow-up lab.

Lab Time

We will begin some very interesting additions to the Physical and Chemical Effects exercises. A table group will expose a agar plate outside the building for the isolation of antimicrobial producing microbes from the air.

Physical and Chemical Effects on Bacteria 49

We will be making some very interesting comparisons. Also we will be checking the antimicrobial activity of the air exposure isolates.

The (fermented) grape juice should be converted to alcohol, 87

which is indicated by the cessation of the CO₂ bubbles and a strong smell of alcohol. We will now have to establish aerobic conditions and inoculate with Acetobacter aceti for the oxidative pathway to vinegar. This will incubate at room temperature until Nov. 22

The Bacteriological Examination of Water 55

Read: 55-59; Reagents 135-137; Biochem & Physio Tests 138. You will be allowed to test your own water if you have a private water supply or water well. You will need to pick up a sterile bottle sometime before this lab. Extra time in lab will be necessary - such as on Wednesday and the following Monday. We will complete the exercise on Nov. 8.

Fungi 63

Read: Linder’s Mounting Solution 135. You will be studying three different aspects of yeast and molds: colonial characteristics, cellular characteristics and physiological characteristics.

Enrichment Cultures - Nitrogen Cycle Microbes 79

Read: Nitrogen Cycle 82-83; Reagents 135-137. This is probably the neatest exercise of the entire semester. We will be looking at the individual steps in the nitrogen cycle as to the microbial involvement. We will initiate the exercise on Nov. 10, by inoculating some specific media with a pinch of soil. We will conclude the exercise on Nov. 22 by chemical testing for all of the involved components.

Viruses 69

We will use two different E. coli viruses (T-1 and T-2) to demonstrate plaque morphology by two different plating techniques. A calculator will be helpful during the follow-up lab.

We will also conclude the Fermentation and Respiration exercise today 87

You will be instructed to examine the “biofilm” growth on the top liquid. Also, you will smell the vinegar.

Preparation and Analysis of Yogurt 73

Read: General Information 51. Students will work in groups of four or by table. You will get try some of this ferment, if you like.

Serologic Reactions 91

Blood typing for ABO and Rh factor grouping. There will be no formal report turned in for this exercise.