I. STUDY OF BACTERIAL CULTURES (Days 1,2,3)

INTRODUCTION
A. Types of Media for Culturing Bacteria:
     The survival and growth of bacteria depends on an adequate supply of nutrients and favorable
growing conditions. In this lab we will investigate some common methods of growing bacteria.
     A solution that contains the nutrients needed by bacteria is called a "culture medium". Basically
all the varieties of culture media can be grouped into two types - the liquid forms called BROTH and
the solid -forms called AGAR. Both media contain the same nutrients, but agar has an extract of

seaweed added to solidify it. Agar is particularly useful in culturing bacteria because of its ability to melt at 100 C, and then solidify again at 40 C. Additionally agar is very good at “displaying” bacteria since most bacteria prefer to grow along the surface of the agar. Lastly, bacteria tend to grow in colonies that vary greatly from species to species.      Before using either agar or broth they should be sterilized in an autoclave or pressure cooker. To STERILIZE means to kill any forms of life already present in the media. This step is done so that only the desired bacteria grow on the media.

     Once the media has been made and sterilized, it needs to be placed into some container. Glass "test tubes" or plastic "Petri dishes" are used to hold the media and growing bacteria. When not in use filled tubes and dishes can be left covered so no bacteria can enter. Tubes and dishes containing bacteria should be incubated. To INCUBATE means to maintain the best temperature for the growth of the bacteria. This is done in a special type of oven called an incubator. During incubation Petri dishes should be placed in an "inverted" position (upside down) so that any water that forms in dish will drain away from the agar.

B. Methods of Transferring Bacteria:
     There are two primary methods of transferring bacteria from a culture onto a Petri dish (also
called an "agar plate"). The first method uses an instrument called a "pipette" that consists of a long
glass tube. Pipettes work much like straws do, by drawing up liquids." Usually pipettes are calibrated
so you can measure how much liquid is being drawn into them. A rubber ball or "pipette aid" is placed on the end of the pipette to draw the liquid up. (NEVER use your mouth when working with bacteria!)

     The second method of transferring bacteria uses an instrument called a "wire loop". It consists of a piece of metal wire inserted into a wooden or metal handle. When the loop on the end of the wire is
placed into a liquid containing bacteria, tiny amounts of the liquid will cling to it. This loop will now leave bacteria on the surface of anything it touches. After each use the loop can be sterilized by placing it in the flame of a bunsen burner in a process called FLAMING.

     Once a tiny drop of bacteria has been placed on a Petri dish it should be spread out. This serves to allow more growth of bacteria, and allows for the isolation of a single colony. A COLONY is an individual mass of bacterial growth on the surface of the agar. All the bacteria in a colony came from one single bacteria, and thus all are identical to one another. Occassionally scientists need to separate out one colony of bacteria in a process called "isolation of a pure colony". The best way to separate bacteria into individual colonies is called streaking the plate. STREAKING means to spread a drop of bacteria across a Petri dish using a wire loop. To properly streak bacteria across a dish follow the instructions listed below. (The following diagram helps explain the procedure)

Streak Plate (Full Version for Isolating Colonies) a. Place a loopful of culture on the agar surface in       area 1. Flame and cool the loop, and drag it      rapidly several times across the surface of area 1. b. Reflame and cool the loop, and turn the Petri dish      90 degrees. Then touch the loop to a corner of the      culture in area 1, and drag it several times across      the agar in area 2. The loop should never enter      area 1 again. c. Reflame and cool the loop, and again turn the dish      90 degrees. Streak area 3 in the same manner as      area 2. d. Without reflaming the loop, again turn the dish 90

STREAK PLATE (FULL VERSION)

STREAK PLATE (S-SHAPED VERSION)      Sometimes all scientists which to do is quickly spread a culture of bacteria across an agar plate to observe growth patterns such as colony size, shape, color, height, odor, etc. A “S”- shaped streak plate is adequate for making these quick observations of bacterial colonies.
	Streak Plate “S”- Shaped Version)

C. Determining Some Bacterial Properties:
A. Cultural Characteristics
     When grown on a variety of media, microorganisms will exhibit differences in the macroscopic (visible by eyesight) appearance of their growth. These differences, called cultural characteristics, are used to help separate identify bacteria. These cultural characteristics can be determined by growing bacteria on agar plates and slants, in nutrient broths, and in nutrient gelatins. In this lab the bacteria will be transferred from nutrient broths to agar plates using aseptic techniques. To properly complete this transfer follow the steps shown in the Diagram #1 below.

Once the bacteria is growing on the surface of the agar plates individual colonies should be located and described using the criteria shown in the Diagram #2 below.

B. Motility Test
     Another test utilized to help identify unknown bacteria is to determine whether it is motile or nonmotile. The technique used in the lab is to grow the bacteria in a semisolid medium (agar) with the bacteria placed INSIDE the media. The agar is used for motility is less dense thus allowing some types of bacteria to move through it. The bacteria are inserted into the media using the “Stab Technique” for motility testing. This technique is illustrated in the Diagram #3 below.

     Once the motility stab is given a day or two to incubate the tube can be checked for cloudiness. If the bacteria is motile, a cloudiness will exist around the stab due to the migration of motile bacteria away from the stab and into the media.
          NOTE -> It is often useful to hold the tube up to the light to look for cloudiness.

NOTE -> The illustration to the right displays a common cause of misreading this test. If the needle is not pulled straight back out of the agar, it will often produce an appearance of spreading bacteria.

C. Catalase Test
     The purpose of the catalase test is to determine the ability of some microorganisms to break down hydrogen peroxide using an enzyme called catalase. During aerobic respiration, microorganisms produce hydrogen peroxide which is dangerous to living cells. If hydrogen perioxide accumulates for long it will destroy the cells, as well and the entire organism. Since hydrogen perioxide is naturally produced as “oxygen-breathing” organisms create energy by burning their food, these organisms have learned to use catalase to break it down. The equation below shows how the hydrogen peroxide breaks down inside the cell.
          2 H O  --------> 2 H 0 = O 
                   2   2                        2          2

To determine if catalase is present in your bacteria follow the procedures shown in Diagram #4 below.

     As the diagram illustrates a colony of the bacteria should be placed on a slide. Then add some hydrogen perioxide directly onto the drop of bacteria on the slide. If the bacteria begins to bubble strongly (gives off Oxygen) it is a Positive catalase test. If there is no bubbling from the bacteria then it is a Negative catalase test.

II. STUDY OF INDIVIDUAL BACTERIA (Days 4,5,6)

INTRODUCTION
A. General Staining Techniques:

     All microbiological staining procedures require preparation of smears prior to the execution of any of the specific staining techniques listed above. The technique, although not difficult, requires adequate care in its preparation. The following basic steps should be followed meticulously.

1. PREPARATION OF GLASS SLIDES: Clean slides are essential for preparation of microbial smears. Grease or oil from fingers on slides must be removed by washing slides with soap and water or
scouring powders, followed by a water rinse and a rinse of 95 percent alcohol. Slides should be dried and placed on laboratory towels until ready for use.

2. PREPARATION OF SMEAR: Avoidance of thick, dense smears is absolutely essential. A good smear is one that, when dried, appears as a thin whitish layer or film. Those made from broth cultures
or cultures from solid medium require variations in technique.
     a. Broth cultures: One or two loopsful of suspended cells should be directly applied to the
glass slide with a sterile inoculating loop and spread evenly over an area about the size of a dime.
     b. Cultures from solid medium: Organisms cultured in a solid medium produce thick, dense
surface growth and are not amenable to direct transfer to the glass slide. These cultures must be
diluted by placing a loopful of water on the slide in which the cells will then be emulsified. Transfer
of cells from the culture requires the use of a sterile inoculating needle. Only the tip of the needle
should touch the culture to prevent the transfer of too many cells. Suspension is accomplished by spreading the cells in a circular motion in the drop of water with the needle tip. The finished smear
should occupy an area about the size of a nickel and should appear as a semitransparent, confluent, whitish film. Before proceeding further, the smear must be allowed to dry completely. Do not blow
on the slide or wave it in the air.

3. HEAT FIXATION: Unless fixed on the glass slide, the bacterial smear will wash away
during the staining procedure. This is avoided by heat fixation, during which the bacterial
proteins are coagulated and fixed to the glass surface. Heat fixation is performed by the rapid
passage of the air-dried smear two to three times over the flame of the Bunsen burner.

     These early steps are universal for all bacterial staining techniques. To better understand these
early steps for making bacterial smears look at Diagram #5 below.

B. Simple Staining:
     The use of a single stain to color a bacterial organism is commonly referred to as simple
staining. Some of the most commonly used dyes for simple staining are methylene blue, basic fuchsin,
and crystal violet. All of these dyes work well on bacteria because they are positively charged.
The fact that bacteria have a slight negative charge causes these dyes to be produce a slight
attraction to the bacteria. This results in these stains sticking to the outside surface (painting) the bacteria.
     The staining times for most simple stains are relatively short, usually from 30 seconds to 2
minutes, depending on the dye. After a smear has been stained for the required time, it is washed
off gently, blotted dry, and examined directly under the oil immersion lens of a microscope.
Since simple stains are not absorbed into the bacteria they do not stain any of the contents of the bacterial Simple stains are useful for determining basic morphology of a bacteria. These
characteristics include the size, shape, and arrangement of the bacteria. The following list of terms
are commonly used to describe the morphology of a bacteria.
          Shape  ->  bacillus = rod-like      coccus = spherical

          Arrangement  ->  individual = found singly      staph = in clusters (grapelike)
                                   paired = found in pairs         strep = in chains

C. Gram Staining:
     Differential staining requires the use of at least three chemical reagents (dyes,alcohol, etc)
that are applied sequentially to a heat-fixed smear. The first step is to add a reagent called the
primary stain. Its function is to impart its color to all cells. Crystal violet commonly serves as the primary stain. This stain dyes all cells purple-blue. As with simple stains, this dye is left on the
bacteria a specific time and then rinsed off.
     The second step is to add a mordant to the primary stain. Its function is to bond to the primary stain thus forming an insoluble complex. The overall effect is to intensify (deepen) the color of the primary stain. Gram’s Iodine is the most common mordant used in Gram staining. All the bacteria will appear purple-black at this point. As with simple stains, this dye is left on the bacteria a specific time and then rinsed off.
     The third step of the procedure is the most critical phase and is called decolorization.
Based on the chemical composition (amount of lipids) in the cell walls of the bacteria, the decolorizing agent may or may not remove the primary stain from the entire bacteria. This step is also the most sensitive ? overdecolorization will always wash away the stain resulting in a false Gram-negative
reading, while underdecolorization will never wash away the stain resulting in a false Gram-positive reading. The timing and procedure for this step must be followed precisely. The most common decolorizing agent is alcohol (95% ethanol). In bacteria alcohol can dissolve the lipids located in the cell walls. Since gram-positive cells have a low lipid concentration only a small amount of the lipid and the
dye coating it is dissolved by the alcohol. As a consequence, gram-positive bacteria do not loose either the primary stain or the mordant and therefore remain blue. Since gram-negative bacteria have a high lipid concentration a large amount of the lipid and the dye coating it is dissolved by the alcohol. As a consequence, gram-negative bacteria loose both the primary stain and the mordant and therefore turn colorless or unstained. As with simple stains, the alcohol is allowed to run over the bacteria for a specific time and then is rinsed off.
     The fourth step involves adding a counterstain to the bacteria. This counterstain has a contrasting color to that of the primary stain. Safranin is most common counterstain used in Gram staining. In Gram-positive bacteria the decolorization step does not wash off the primary stain, therefore the
safranin cannot be absorbed. Thus Gram-positive bacteria will retain the color of the primary stain. In Gram-negative the decolorization step does wash off the primary stain, therefore the safranin is absorbed. Thus Gram-negative bacteria take on the color of the safranin and turn reddish-pink.
     Gram staining is an example of a differential stain. Such stain can distinguished specific traits between bacteria. Gram stains for example can indicate the cell wall structure of bacteria, and thus
can be used to predict certain behaviors of these bacteria (ie. reaction to specific antibiotics). The
Gram Staining process is named after Dr. Christian Gram. Because it divides bacterial into two major groups, gram-positive and gram-negative, it serves as an essential tool for the classification and differentiation of microorganisms.
     To see an illustration of the Gram-staining process look at Diagram #6 below.

III. IDENTIFICATION OF UNKNOWN BACTERIA      (ALL Days)
     Look at the data you’ve collected concerning the unknown bacteria. Use the table shown below along with those characteristics you’ve collected to find which bacteria listed in the table “BEST FITS” the description of the unknown bacteria. (NOTE ? It is not uncommon that none of the bacteria in the table below exactly fits the unknown // Some of the tests conducted are very subjective)

Bacteria Identification Expected Results