Medical Technology

Medical Technology

Microbiology Lab Manual

Isolation and Identification of Bacteria

from Mixed Bacterial Cultures

for Medical Diagnoses

Laboratory Notebook Record Keeping Procedures

Use only your official microbiology notebook to record your work. All work must be recorded in the notebook and in no other document.

Date and sign the top of every page.

Sign and date at the end of each experiment.

Number every page. (For consistency, the entire class will number the outside corners of their lab manual.

Do not tear out any page(s).

Maintain a Table of Contents as you make entries in the notebook. The first page of every lab investigation should be listed in the Table of Contents.

Make all entries in black permanent ink. No pencil entries are permitted. The use of colored pens or pencils is acceptable in some cases, as approved by the supervisor.

Neatness is mandatory. All entries must be legible, in your neatest writing, such that a judge or a court of law can read, comprehend, and verify your data or original thought. Use a straight edge (rulers) to create tables and graphs. Skip lines, as necessary, for clarity, so as to not crowd information.

Do noterase, ink over, or white out errors. Draw a single line through errors so they can still be read. Place your initials and the date next to the correction.

State the objective or purpose of each experiment, and reference previous work or projects.

To protect the credibility of the notebook, there should never be any blank pages or blank spaces below an entry. Blank pages need to be crossed out and initialed and dated. Blank areas under images, tables or graphs should be labeled “NWUI – or Nothing Written Under Insert” then initialed and dated.

Blank or unused pages can be labeled, “ILB – or Intentionally Left Blank” then initialed and dated.

Use “from…” or “go to…” statements to tie together sections of a lab report of continuous work. When procedures, data, or conclusions, etc., are continued from previous pages, each page must have a “from page___” listed. When continuing to another page, there should be a “go to___” statement directing the reader to the next page of that work.

Record all procedures, materials, and quantities used, plus reactions or operating conditions, in sufficient detail and clarity so that someone of equal skill could understand or repeat the procedure if necessary.

Avoid abbreviations and codes when possible. Only the standard abbreviations from metric measurements may be used universally. Any coding or special labeling on samples or in procedural notes should be fully recorded and explained in the notebook.

List all persons from whom samples or data were obtained, shared or transferred.

Attach as much original data as is practical in the notebook. Where it is not practical to attach original data, attach examples and make clear reference to where the original data are stored.

For important entries, such as key conclusions or new ideas, have a coworker sign and date the entry. Be sure the coworker is not a co-inventor, but someone who is capable of understanding the meaning of the notebook entry.

Write/print clearly so there is no ambiguity about the information recorded. Skip lines between data tables, graphs and important conclusions to make it easier to find and read recorded information.

All data, especially tables, pictures, and graphs, must be properly titled, labeled as necessary, and annotated. Indicate scale and/or magnification, as necessary, if drawing or taking pictures of organisms from a microscope. Annotations must explain what the data is about.

Follow proper scientific rules for drawing. (refer to your physiology notebook).

All data must be witnessed, dated, and signed by a coworker that is not a co-inventor.

Pre-Lab Requirements

It is an expectation that you come prepared for the labs during the microbiology series. Labs must be read and pre-labs completed before you are granted permission to continue.

Pre-labs are to be completed prior to each lab.

Regular lab entries will be completed on a new page following the pre-lab.

Title the pre-lab with descriptor:
Ex. Pre-Lab 1 – Aseptic Technique and Inoculation of Medium

Restate in your own words or copy the purpose into the pre-lab section.

Read the “Background” section of your lab manual, and highlight directly in the lab manual. Then OUTLINE the background information for your pre-lab. Make sure the outline is of “appropriate” length.

Read and highlight the procedure directly in your notebook. (Mark the text). Then proceed by drawing a flowchart of the procedures. The flowchart should be neat, clear, and concise, with annotated notes of the steps, as shown to the image on the right. If you are not familiar with a piece of equipment or of a technique, you may need to look it up on the internet to better understand the techniques so that you can do a proper flowchart.Make the flowchart images large and clearenough, such that a fellow coworker can replicate the procedures simply by using your flowchart. Chicken scratch will not be accepted.

When applicable, create a “rough draft” of the kind of data table you will need to organize your results. You will share this data table with your group, and the data table that your group finally decides on may be different. You may not always need to create a data table for each lab.

Lab 1: Aseptic Technique and Inoculation of Mediums

Purpose:

To practice the safe, aseptic transfer of bacterial cultures between different mediums.

Background:

In nature, microorganisms exist as mixed populations of many different types. However, our knowledge of microbiology has increased through the study of isolated species, grown in environments free from contamination by other living organisms such as fungi.

It is necessary for you to gain the skills to successfully transfer a pure culture of bacteria from a stock culture source to a new, sterile medium and to do so without inoculating it with any contaminating bacteria. This skill is known as aseptic technique.

In the inoculation procedure, a metal inoculation loop is used. You will be initially transferring from a stock, broth solution into a new sterile broth tube. After this transfer, you will be transferring from the stock broth to a solid, agar medium in order to grow isolated colonies. In each of these techniques, the sterility of the inoculation loop as well as even the air in and around the culture tube is crucial. Flame will be used to sterilize the inoculation loop by heating it until it is red hot, then allowing it to cool. Laying down, waving, or blowing on a cooling loop will contaminate it. The surface over which you are working must be sterilized before and after you do this lab and never should an open container of bacteria be laid down without proper closure nor the loop set down again before it is sterilized. Leaving a culture tube or agar plate open to the environment exposes it to bacterial spores which only need to land on its surface to colonize and contaminate your cultures. Be mindful of what you are doing!

Safety/Disposal:

In order to maintain a sterile environment, and to maintain a level of personal safety, do not eat or drink during labs. All lab stations will be cleaned with a 10% bleach solution, and you will need to wash your hands, using the guidelines from the World Health Organization. Cleaning of lab stations and washing of hands will be completed before and after each lab. Long hair must be tied back. Hair is one of the top contaminates in a microbiology lab.

Be careful when working with the inoculating loops as they can be very hot and will cause burns. Never point the burners towards people or yourself.

Dispose of materials as instructed. Bacteria are never disposed of in the trash, unless it has been heat-treated and killed through an autoclave process (or killed using a bleach solution). Wipe down the lab table with 10% bleach solution and wash hands thoroughly before leaving the lab.

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Materials for each lab group:

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Lab stove

Sharpie

Eight nutrient broth tubes

Inoculating loop

Test tube rack

Stock Solutions:

Bacillus sp.
Staphylococcus epidermidis
Enterococcus faecalis
Pseudomonas aeruginosa
Escherichia coli
Serratiamarcescens

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Procedure:

Obtain 7 tubes of nutrient broth.

Your group will inoculate each of the tubeswith a separate stock culture or as per assigned by the teacher. The last tube will be your control tube. Label your tubes with the name of the bacterial culture, date, initials, and station number. (For example: Bacillus sp. 1/19/13MA-1) Label the control tube as Control. Include the date, your initials, and station number on the control tube as well.

Start with the control tube. Practice aseptic technique by first flaming the loop. Remove the cap of the tube using the procedure demonstrated (Figure 1) and flame the mouth of the tube. Insert the loop as to “inoculate” the broth. Re-flame the mouth of the tube and recap it then re-flame your loop. With good technique, this tube should not have any growth. Each member of your group will repeat this procedure with the control tube.

The other tube should be inoculated with the bacteria. Flame the loop, letting it cool. Place both the stock culture and sterile tube in your left hand. Uncap both, and keep the caps in your right hand. Dip your cooled loop into the stock culture and carefully pull out a loop of the culture, carefully putting it into the sterile tube. Re-flame the tube openings. Replace the caps and re-flame your loop. Each person in your group will repeat this procedure.

Repeat the above procedure (step 4) with the remaining five stock cultures.

Incubate all seven of your tubes in the incubator at 37 degrees Celsius until next class, putting the tubes in the class test tube rack.

Lab 2: Aseptic Streaking of Agar Plate for Isolation of Colonies

Purpose:

To isolate bacteria from each other on an agar plate into individual colonies

Background:

In order to separate one type of bacteria from another and to view individual colonies, it is necessary to spread a broth culture very thinly over the surface of agar plates so that they can be separated enough to be distinguished individually. This is called the streak plate method. The objective of this procedure is to separate bacteria from each other so that they can be viewed as individual colonies. Each colony begins as one bacterium. So the separation of a small amount of the culture is crucial. These cells will start packed as a drop from the inoculation loop. As the streaking occurs, the cells are spread out farther from each other, allowing individual colonies to grow.

You will need to make very careful observations of the resulting plates you inoculate from each of the stock cultures, as you will be separating mixed cultures using two or three of these stock cultures and attempting to purify the mixture in a few weeks (ONLY IF TIME PERMITS)

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Materials for each lab group:

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Lab stove

Sharpie

Six nutrient agar plates

Inoculating loop

Stock Solutions:

Bacillus sp.
Staphylococcus epidermidis
Enterococcus faecalis
Pseudomonas aeruginosa
Escherichia coli
Serratiamarcescens

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Procedure:

Your group will make one agar plate for each of the stock culture. You will use nutrient agar plates for all of the cultures. Before you inoculate, label the bottom of each plate (along the edges) with your table number, name of the bacteria you will use, and the date.

Flame the inoculating loop and allow it to cool. Remove the cap of the stock culture and flame the opening.

Insert the loop into the tube and pull it out slowly. There should be a film of broth inside the loop. If it pops, try again.

Re-flame the mouth of the tube and recap it. Return it to the rack – you might want a partner to help you with this as you are carrying a loop filled with a bacterial culture.
Carefully lift of the lid of the plate. Do NOT place the lid with the rim facing down on your working surface. This will contaminate your lid. If you need to set your lid down, set your lid down, rim facing up.

With your loop, gently mark a line on the agar, from the top of the agar plate moving down the midline, approximately 1 cm. This will note the place that you initially deposited your culture. Be careful not to gouge the surface! (This is crucial!)

Place the loop outside of the starting midline to the left and move the loop across the midline to the right. Continue streaking for about 1/3 of the plate. See image above.

Rotate the plate a 1/3 turn. Flame and sterilize your loop. Place the loop outside the streaked area. Move through the streaked area one time then continue streaking away from the first area. Streak for 1/3 of the plate, being careful not to cross (move) the loop through the original streak.

Rotate another 1/3 of a turn, flame your loop, and repeat. This will create individual colonies since you are essentially spreading out the bacteria and isolating one bacterium from another.

Replace the lid, re-flame the loop and let it cool.

Tape the lid shut. You will be incubating the inoculated plates, lid-side down, for 24-48 hours. Note the incubation temperature of 37 degrees Celsius in your lab manual.

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Lab 3: Observation of Cultures & Colony Morphology

Purpose:

To practice observing broth and agar cultures for evidence of growth, as well as to learn to classify colonies by specific descriptors.

Background:

Bacteria have exceedingly high rates of binary fission and are therefore easy to culture in a lab. Evidence of growth in a broth medium will be seen as a cloudy appearance in a previously transparent solution. The bacteria will tend to form a pellicle at the bottom of the tube and can be suspended by vortexing or careful mixing.

Agar colonies start with only one bacterium! Each dot on the agar is a colony consisting of thousands or millions of clones of the original bacterium. Each species produces colonies that have characteristics specific to that species. By careful observation, you should be able to differentiate between individual colonies.

Procedure A: Observation of Broth Cultures

1. Examine each tube from Lab 1 for evidence of growth. Evidence of growth would appear as cloudiness (known as turbidity), a collection of material at the bottom of the tube (a pellicle) or along the top of the tube. A tube that has no growth would remain clear and transparent.

2. Create a table for your results. Make sure to note the organism that was inoculated in broth and the date. Note the results and color of the culture of both the control and the experimental tube in your lab notebook. Make necessary observations. Be clear in your data, observations, and drawings. Don’t forget to get a witness for your data!

Procedure B: Observation of Streaked plates

1. Maintain careful records of the observations of each streaked plate. You need to be organized with your records and detailed in description. Create an appropriate data table to accomplish this.

2. Observe the plates looking for individual colonies. These spots should be isolated from each other – not touching.

3. Use the following descriptors in your data table to record in detail about the appearance of the colonies so that you can use this information when you work with your unknown, mixed cultures. Include an extra column in your data table for remarks or additional observations that you may want to note about each species of bacterium.

Descriptors of Colonies:

A. SIZE OF COLONY (measure with a millimeter rule), less than 1mm = punctiform (pin-point).

B. EDGE/MARGIN OF COLONY: See image below

C. COLOR: white, buff, red, purple, etc.

D. OPACITY OF COLONY: transparent (clear), opaque, translucent (almost clear, but distorted vision–like looking through frosted glass), iridescent (changing colors in reflected light)

E. ELEVATION OF COLONY (turn the place on end to determine height)

F. SURFACE OF COLONY: smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled)

G. CONSISTENCY: buttery, viscid (sticks to loop, hard to get off), brittle (dry, breaks apart), mucousy

H. ODOR: Absent or present? If it has an odor, what does it smell like?

**Some characteristics are easier to see and note than others. If a characteristic cannot be determined, do not make-up data. Simply mark your data table accordingly. For example, “not discernible”, or ND.

Lab 4: Bacterial Staining Methods

Purpose: