Safety Precautions:

1. Always use STERILE TECHNIQUE

2. ASSUME that any bacteria that grows on your plate is harmful and treat as such

3. Always WASH HANDS after you are finished handling plates

Materials

Prepoured Agar Plates

Sterile Swabs
Innoculating Loop

Known Cultures, Gram + and -

Gram Stain Kit

Microscopes

Bacteria Lab

A solid source for bacterial growth is essential if organisms are to be isolated. In the laboratories of Robert Koch, gelatin was first used to achieve bacteria colonies. Agar now serves as a more useful material; it will remain solid until heating and then will solidify as it cools. Before it cools (and solidifies) it can be poured into petri dishes. Using sterile technique minimizes the contamination of the agar before you are ready to inoculate it with bacteria from other sources.

In this lab you will be innoculating plates and observing bacterial growth. Microscopes can then be used to identify specific bacteria. This lab may take several days, keep all data and observations in a separate notebook to be compiled and organized into a final lab report.

I. Preparing and Innoculating Your Plates

Obtain (or make) an agar plate. It is important to not contaminate the agar before you are ready to take your samples, so leave the lid closed. Microbiologists always label the bottom, not the lid, of the plate so that the lids do not get accidentally mixed.

Without opening any of the petri dishes, use a marking pen to divide the dish into four quadrants and label the sections as shown on the bottom of the plates.

Surface 1: Any surface in classroom, hall or bathroom.  Use sterile cotton swabs.
Surface 2: Any surface in classroom, hall or bathroom. Use sterile cotton swabs.

Hair: Place a small length of your hair in this quadrant
Finger: Touch the circle marked D with your finger.  Then wash your hands and rub your finger with alcohol.  Place your “disinfected” finger in circle C

II. Analyzing Colonies

You will identify and categorize different bacterial colonies based on varied appearance and morphology (form and structure), When a single bacterial cell is deposited on the surface of a nutrient medium (agar), it begins to divide exponentially. After thousands of cells are formed, a visible mass appears which is called a COLONY. Each species of bacteria will exhibit characteristic colonies.

Terms

1. Draw each plate, showing how colonies are spread across the agar surface.
2. Pick several colonies on your plates and describe them using the terms above.
3. (Optional) You may wish to photograph your plates and include them in your final lab report.

III. Preparation of a Bacterial Smearloop

1. Place a drop of water on a clean slide. check box
2.  Heat the inoculating loop until it glows red.  Let it cool then remove a small amount of culture from the agar surface; touch it several times to the drop of water until it just turns cloudy. check box
               More is not better! Too much and you won't be able to see individual cells
3. Burn the bacteria from the loop and allow the loop to cool, use the loop to spread the suspension (water + bacteria) over the surface of the slide to form a thin film.check box
4. Allow suspension to air dry.  This process will be short if you spread the liquid out in step 4.  check box
5. Hold the slide with a clothespin and then heat fix the bacteria on the slide by passing it over the flame 3-4 times (film side up). Do not overheat the slide, it should feel warm but not hot. check box
6. Place a drop of methylene blue on the slide over where the “smudge” of bacteria and water is
7. Allow the dye to remain for approximately 1 minute (the bacteria will take up the stain during this time) check box
8. Wash the excess stain off the slide by picking the slide up and holding it at an angle over the sink. Gently rinse the excess dye off with water. check box
9. Blot off excess stain and water using filter paper - DO NOT RUB! check box
10. Examine the slide under the microscope using the high power objective. You do not need a coverslip. check box

You may need to repeat this process a few times in order to get a good slide!

Draw your bacteria; identify the shape and arrangement of the bacteria. You may wish to look at preserved slides at this time to compare to your own samples.

IV. GRAM STAIN

Microbiologists use a special level of classification to describe eubacteria – it is called the “Division”.  Eubacteria are placed in a division depending on the type of cell wall they have. A bacterium with a cell wall that has a large amount of petidoglycan, is classified as gram (+) and stains blueish-purple.   Bacteria with a thin peptidoglycan layer are classified as gram (-) and will stain pink.

Gram stains can be difficult and take practice. Follow the steps exactly as listed. If done correctly, you should be able to identify your bacteria as Gram + or Gram - and check resouces to see if you are correct. If known samples are not available, you can gram stain a sample from your petri dish, but there will be no way to check your accuracy.

1.  Add a sample of a known culture to your slide and pass it briefly over flame to heat fix the sample.check box  
2.  Add 5 drops of crystal violet over the fixed culture.  Let stand for one minute.check box
3.  Pour off excess stain and gently rinse the slide in tap water.  Keep in mind, the objective is to wash off the stain, not the fixed culture.check box
4.  Add about 5 drops of iodine to the smear.  Let stand for one minute.check box
5.  Pour off excess iodine and rinse the slide with water as before.  check box
6.  Tip the slide and add several drops of the ethyl alcohol to the upper end.  Allow alcohol to flow over the smear.  After several seconds, rinse the slide again. check box
7.  Add about 5 drops of the safranin to the slide.  Let stand for one minute.  check box
8.  Rinse the slide again, drain and blot away excess moisture.  Let the slide dry. check box

Compare each of the samples colors, taking note of whether the sample a light pink color (gram -) or a dark purple color (gram +). Don't forget to write down the names of your samples to include in your lab report later.

Bacteriology – Lab Report Guidelines

1. Title (Something clever)

2.  Purpose – Describe the objectives of this activity.

3.  Sections

  1. Preparing your plates (discuss the samples you took, how your colonies appeared, differences in the quadrants, how the plates were prepared and what nutrient was used to grow the bacteria
  2. Analyzing colonies (discuss the types of colonies you observed, use appropriate terms to describe the colonies, include a photo or sketch)
  3. Preparation of a Bacterial Smear (compare your bacteria colonies to preserved slides, discuss difficulties you may have had observing individual bacteria, describe what you saw under the microscope)
  4. Gram Stain (explain the purpose of a gram stain and how it is different from the stain you did in section 3, describe the procedure, discuss any difficulties or errors that may have occurred during the procedure, identity whether your bacteria was gram + or gram -)

4.  Conclusions – What did you learn?   What would you like to do over or expand upon?  This is the area for your own reflections and thoughts.

Must be TYPED.  Due Date: _________