MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #3: SIMPLE STAINING AND STREAKING

NAME, ID #:_______________________________________________

NAME of TA: ______________________________

REVISED: 08/23/99

INTRODUCTION TO EXERCISE:

SIMPLE STAINING

As you've observed by viewing wet mounts in the first four exercises, most bacteria LACK COLOR or CONTRASTING INTERNAL STRUCTURES. Since the density of most bacteria is only slightly greater than that of water, bacteria are difficult to see in liquid suspension using a bright-field microscope because they are almost transparent. To visualize bacteria, dyes or STAINS are used. In some ways staining is similar to what many of us did to Easter Eggs as children. Bacteria normally carry a negative charge (-). Most bacterial stains are either POSITIVELY (+) or NEGATIVELY (-) charged, but some stains carry no charge (0). Positive stains are ATTRACTED to the negative bacteria, whereas negative stains are REPELLED from the bacteria. A few stains (e.g. India Ink) are composed of particles too large to enter the bacteria and they have also been called negative stains. Staining not only makes bacteria more visible, but it allows their morphology (e.g. size and shape) to be visualized more easily. Visit this site and examine the stains of several bacterial under the RESULTS section.

To prepare bacteria for staining (Fig. 1 & 2) a sample of bacteria in liquid is spread as a thin layer or SMEAR on a clean glass slide. The thin bacterial film is allowed to AIR DRY and then it is FIXED to the glass to prevent it from washing off during the subsequent staining procedure, by passing the slide briefly through a flame; this treatment sticks (glues) the bacteria to the glass and also kills any pathogens. The fixing process in similar to what happens when you FRY AN EGG and the egg-protein sticks firmly (FIXES) to the pan. The bacterial film should not be too thin or too thick. If it dries as a THICK OPAQUE LAYER it is too thick and you should DISCARD such smears and start again. Conversely, if you can't see ANY CLOUDINESS on the dried slide it may be too thin. Further, your slide must be CLEAN and FREE OF OIL from your fingers or the bacterial film will not stick. Once you have a fixed bacterial film it is flooded with the respective stain and allowed to sit for approximately ONE MINUTE, during which time the stain PENETRATES THE CELLS. The stain is then washed off with a gentle stream of water and BLOTTED DRY. The dried, stained smear is then viewed with the microscope. Your instructor will demonstrate these techniques.

STREAKING FOR ISOLATION

Bacteria rarely exist alone in nature. A collection of a single type of bacteria isolated and growing free from all other microbes is called a PURE CULTURE (PC). Microbiologists almost always study PC of bacteria because it is the only way to learn about the morphology and physiology of individual bacterial species. The most common technique to obtain a PC involves RUBBING A MIXTURE of bacteria across a solid surface of some material that the bacteria will grow on. Usually this is a sterile plate of bacterial MEDIUM (FOOD) that has been solidified with a material made from seaweed. This solidifying material is called AGAR AGAR (this is not a misprint, however we will refer to it simply as #AGAR from now on).

The streaking technique consists of picking up a bacterial mixture on the end of a STERILE WIRE LOOP and rubbing it rapidly across approximately 1/4 of the surface of a STERILE AGAR-PLATE. The loop is then sterilized, cooled and streaked at approximately a right angle beginning in the end of the first streak. Again approximately 1/4 of NEW surface is covered by the second streak. The process is repeated twice more, each time rubbing the freshly sterilized loop into the end of the previous streak to pick up a few cells and streaking these cells into a new, virgin area of the plate. In this way the various microbes in the original mixture are randomly rubbed off of the loop and deposited as SINGLE CELLS onto the solid surface of the medium (Fig. 4). The INDIVIDUAL cells then grow and produces a BACTERIAL COLONY (a visible mass of bacteria) which is a PURE CULTURE. Actually, a microbiologist rarely trusts the first streaking to give them a PC, so they pick an ISOLATED COLONY from the first streak plate and REPEAT the streaking process once or twice more to be absolutely certain that they really have a PC. Your instructor will demonstrate this technique. You will then streak a mixed culture on an agar-medium plate. Following colony growth you will examine some of the possible pure culture colonies.

PURPOSE OF LABORATORY:

To learn how to prepare a BACTERIAL SMEAR.
To learn how to stain a bacterial smear.
To learn how to use the oil immersion lens.
To determine the form, shape, size and arrangement of stained bacteria.
To learn how to purify or separate bacteria from each other using the STREAKING TECHNIQUE on solid AGAR PLATES of bacterial MEDIUM.

RELATIONSHIP TO LECTURE MATERIAL

NetText101/102: CHAP. I; Early microbiologists.

GENERAL INSTRUCTIONS:

Draw and label your observations in the appropriate circles below.
Be sure that you understand ASEPTIC TECHNIQUE before proceeding.
Be certain you understand the theory and mechanical process of STREAKING. Do some dry runs on the lid of your petri dish to get the feel of streaking.
Click here or here to view a Quicktime movie, with sound on the aseptic transfer of bacterial culture material. This movie takes about 5 to 20 min to load depending on your Internet connection.

PROCEDURE:

STERILIZATION OF A LOOP BY FLAMING

Figure 1. Flaming of loop. Place the loop in flame starting at the loop and move it through the flame so that the wire becomes red-hot along 4 to 6 cm of its length. Allow the wire to cool for about 10 seconds. Pick up the sample with the cooled loop and distribute it. Repeat the flaming/cooling procedure before laying the loop down on the desk. Always flame the loop immediately prior to using it for any purpose. Click here to see a picture of loop being flamed. At this site there is a complete series of pictures of the basic processes of aseptic technique; View them ahead of time as the TA may ask you to describe the physical appearance of the person in the pictures. This site has a Quicktime Movie on aseptic technique.

PREPARATION OF A BACTERIAL SMEAR

Figure 2. Preparation of a microbial smear for STAINING. For a liquid sample, use a sterile/cooled loop to pick up some liquid sample in the loop. Place this liquid on the slide. For a dry sample, use your sterile/cool loop to first put a small drop of water or sterile medium on the slide. Then pick up a tiny bit of the microbe sample from the source (colony, wound) with a sterile/cool loop and mix it into the liquid on the slide, being careful to not put too much or too little sample on the slide. Spread the sample-drop for drying and fixing as illustrated above and as demonstrated by your instructor. Click here to see a smear being done.

STAINING

Figure 3. Adding stain to the fixed bacterial smear. After the bacterial smear has been heat-fixed to the slide, lay it over the sink on the slide-support. Carefully drop the appropriate staining solution onto the smear so as to cover it entirely. Allow it to sit for 30 to 60 seconds. Then tip the slide so the excess stain drops into the sink. Then gently run tap-water or a spray of deionized water over the smear, washing off any remaining stain. Finally, dry the smear either in the air or by gently patting it with absorbent paper. Examine under the microscope, first using the 10X to locate areas of stained material, then place a drop of oil on the dried, stained sample and rotate the oil-immersion lens into the oil drop.

GENERAL INSTRUCTIONS

Have your Rodac plates available. If you haven't incubated them do so now, otherwise place them in your drawer for lab #4. The TA will randomly ask to view the results of your Rodac plate.
Before beginning this procedure read Section 3 in A photographic atlas for the microbiology laboratory. Examine the pictures of the various stained bacteria (pg. 21-26) in the following links: Bacillus; various clinical stains; many stained bacteria. At the conclusion of this experiment your stains should resemble those shown in the Atlas.
Samples for this experiment:
- For Simple Staining
  - Cultures of appropriate bacteria (e.g. Escherichia coli, Staphylococcus aureus, Micrococcus Lutea, Bacillus subtilis and Enterococcus.
- For Streaking
  - Cultures of Escherichia coli and Bacillus subtilis and, if required, an appropriate mixture of two bacteria to test your streaking abilities.
- Rodac plates:
  - If there is sufficient time you are encouraged to stain microbes found on your Rodac plates.
- Hay infusions, pond water etc:
  - If there is sufficient time you are encouraged to stain microbes found in the various water samples provided for the first 2 weeks of labs. The film floating on the liquid usually proves to be especially interesting.
Prepare the smears for staining as in Fig. 2 and as demonstrated by your instructor.
Stain the dried and fixed smears with the appropriate dyes as described below.
- Label and save any stained smears you didn't examine in this lab for viewing at a later time.

NO ONE CAN LEAVE CLASS EARLY

SPECIFIC INSTRUCTIONS

Divide three clean slides (one for each stain used) into three sections by drawing lines or circles on the bottom of the slide with a wax pencil.
- Suspend the cells from a colony on the Rodac plates in drops of water on one of the sections of your first slide as demonstrated by the Instructor and in Fig. 2.
On the first slide stain (1) a colony from a Rodac Plate; (2) a sample from the pond water; (3) a sample from the film on the top of the hay-infusion culture. Stain this slide with the Crystal Violet.
ASEPTICALLY remove loops of bacterial suspension from the respective pure cultures and spread them out in ~1 cm circles on the divided slides.
- Record which bacteria/samples (e.g. Pond water etc.) and stain are on each smear. The partners are to stain all of the pure strains between them with all three dyes and to view each other's stains when completed.
- Stained slides may be stored in the desk and viewed at the next lab.
Allow the bacterial suspensions to dry in the air while you are streaking the plates.
- Follow the instructions and streak the samples indicated. Be sure to label the bottom of the plates with name, date and organism or sample.
After streaking the plates heat-fix the bacteria on each of the slides, as demonstrated by the instructor. Do NOT OVERHEAT or you will end up with tiny charcoal briquettes. Allow the slides to cool 30 to 60 seconds.
Add one of the different stains to each of the heat-fixed slides; record which stain was used on each slide. After approximately 30 seconds to one minute, wash off the stains with a gentle flow of water.
Blot dry and examine with the microscope, starting with the 10X and moving directly to the 100X oil immersion lens.
Draw and label each stain as to sample, stain and magnification in the circles below.
Describe the cells in the pure cultures as to their FORM, SHAPE, SIZE and ARRANGEMENT of cell grouping (view the next page also). For example, a description might be: short, thin rods, lying in pairs or singly, few long chains etc.
Describe the stained contents of the others cultures/samples as to the predominant cell types under your drawing of same.
Visit the site in #8 and this site and this site to compare your stains with those of the same species of bacteria that you stained.

Figure 4. Illustration of streaking for purification. The colors represent the pattern of growth (of the same bacterium) seen in each respective streak-zone.

STREAKING FOR ISOLATION

Before doing this exercise read section 2, pg. 9 in A photographic atlas for the microbiology laboratory and examine the figures on pg. 1-4.
Practice streaking on the open palm of your hand or with sharpie on a circle on paper the size of a petri dish.
Sterilize/cool your loop (Fig 1).
Dip the sterile loop into the culture and withdraw a loop of the suspension.
- One partner will streak Bacillus subtilis and the other E. coli (or other appropriate bacteria or mixture of bacteria as instructed).
Remove the upper lid of your petri dish and place it upside down on the table.
Hold the loop parallel to the agar surface and lay it gently on the agar, tipped slightly up, about 0.5 cm from the edge of the plate and, without lifting the loop, wipe it lightly back 'n forth across the agar surface while drawing the loop slowly across ¼ of the plate; each sweep of the loop should be in a new or virgin area of the agar. Continue until you have streaked about ¼ of the agar surface (Fig. 5 #1).
Sterilize/cool the loop.
Rub the sterile loop across the END of the previous series of streaks two or three times to pick up a few microbes. Then begin streaking as before into a fresh area of the plate. Stop when you have covered another 1/4 of the plate's surface (Fig. 4 #2).
Repeat steps # 5 & #6 two more times, at which point there should be 4 streaks on the agar surface as shown in the illustration above and 85 to 95% OF THE PLATE SURFACE SHOULD BE STREAKED.
Label the bottom of the plate with your name, the sample name and date and place it UPSIDE DOWN in the incubator or in your desk drawer according to your TA's instructions.
At the next lab. compare your plates with those in A photographic atlas for the microbiology laboratory. Assess your technique (A to F) and consider how it can be approved if necessary; See if you obtained isolated colonies. See if your TA agrees with your grade.

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

How would a positively charged stain (dye) react when mixed with the average bacterium?
How does the streaking process work to produce a PURE CULTURE?
Why do you flame the loop by holding it ABOVE the flame?
Why do you invert the petri dish when incubating it?
Why do you flame the opening of a test tube or flask when removing or adding microbes?
Draw and label the major shapes/forms/arrangements of bacteria.
What does "heat fixing" do?
Who discovered the usefulness of agar for growing bacteria?

Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
SCIENCE HALL, ROOM 440CA
PHONE: 509-335-5108
E-mail address: hurlbert@wsu.edu or hurlbert@pullman.com
OFFICE HOURS: Mon. Wed. 2:00-4:00 PM

TOC