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

Investigation: Bacteria

Essential Question: How Can Bacteria be Studied?

Goals:

check box 1. Inoculate plates with samples taken from surfaces at the school and observe growth of bacteria colonies.
check box 2.  Use microbiology terms to describe the appearance of colonies and compare samples / surfaces.
check box 3.  Use a microscope to examine bacteria from colonies.
check box 4.  Examine prepared slides of bacteria and compare different strains with regard to appearance and location.
check box 5.  Perform a gram stain using a step-by-step guide to compare gram negative bacteria to gram positive bacteria using known samples.
check box 6.  Use sterile technique and observe all safety protocols when handling 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.

A. Inoculating Your Plates

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.

Your agar plates will be prepared for you in advance.

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.

bacteria plate

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

D: Touch your finger gently to this circle.
C: Clean your finger with an alcohol pad or soap and touch it to this sector.

B. 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.

C. 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!

*Identify the shape and arrangement of the bacteria. Make sketches of each to include in your final lab report.

D. Observe Known Bacteria samples

Use internet sources, slide viewers, and prepared slides to identify the different shapes and types of bacteria. Your investigation should include, but not necessarily be limited to the following well-known bacteria.

1. Escherichia coli
2. Staphylococcus aureus
3. Bacillus subtilis
4. Cholera vibrio

*Include sketches and descriptions in your final lab report as well as an overview of the bacteria. This is something you may need to research on your own.


E. 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 dark-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 resources 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. Use a sterilized innoculating loop to transfer a small droplet of water to a clean slide. Sterilize loop. check box
2.  Add a sample of a known culture to the droplet and pass it briefly over flame to heat fix the sample.check box  
3.  Add 1-4 drops of crystal violet over the fixed culture.  Let stand for one minute.check box
4.  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
5.  Add about 1-4 drops of iodine to the smear.  Let stand for one minute.check box
6.  Pour off excess iodine and rinse the slide with water as before.  check box
7.  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
8.  Add about 5 drops of the safranin to the slide.  Let stand for one minute.  check box
9.  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.

If your gram stain was not successful, use google to research your sample and find out what it is.


Bacteriology – Lab Report Guidelines                                                    Must be TYPED.  Due Date: _____

Review the "Goals" listed at the beginning of this document. Your job is to demonstrate that you have achieved these goals.

Choose One Project for your Final Report

Infographic 

Create an infographic (piktochart), Slide Presentation, or traditional lab report  (typed) that demonstrates that you have achieved the GOALS listed at the beginning of this lab guide.   You are free to use whatever means you think will show me that you have engaged and learned something in this lab.  Suggested content:

Photographs (your own)
Images from websites (cite sources)
Paragraphs / Essays / Lists that explain a procedure or understanding (please do not copy this lab guide, summarize or put in your own words)
Drawings of samples

Must be TYPED.  Due Date: _________

You will be graded on your demonstration of achieving the goals listed in the beginning of this lab guide.

Grading Rubric

Aseptic Technique observed during lab, locations where samples were taken described __3__2__1__0
Growth of colonies on plates observed, described in detail using appropriate terminology __3__2__1__0
Individual bacteria observed using a microscope, descriptions included __3__2__1__0
Gram stain procedure attempted, student was able to stain and identify types of bacteria __3__2__1__0
Overall Presentation - report shows attention to detail, provides evidence from the lab, organized __3__2__1__0