Introduction

Bacteria are the most numerous and, arguably, the most successful organisms on the planet. They are found virtually everywhere. Their diverse sources of nutrition, rapid rates of reproduction, and adaptive abilities have enabled them to survive successfully in almost every habitat. From the arctic tundra to the near-boiling hot springs, from the depths of the ocean to the internal organs of other living things, bacteria have thrived since the very earliest beginnings of life on Earth.

Bacteria are in the Kingdom Monera. Monerans are prokaryotes–organisms that have a nucleoid and mitochondria. They have DNA chromosomes and are very rarely multi-cellular. Most bacteria are very small, ranging in size from 1– 10 microns, and their metabolism is highly variable, unlike the “standard” metabolic oxidation modes of higher organisms.

There are two major groups in the Kingdom Monera: the Archaebacteria and the Eubacteria. As the name suggests, the Archaebacteria are an “ancient” form that evolved from the bacteria that first existed several billion years ago, when conditions were not conducive to any other type of life. Archaebacteria are unique in their chemical makeup and the structure of their DNA and include the methane-producers that break down organic matter and produce methane gas. “Salt-loving”, or halophilic, bacteria that inhabit the Great Salt Lake and the Dead Sea are another group of Archaebacteria. A third group of Archaebacteria is adapted to thrive in the waters of hot springs at temperatures up to 90°C. This group has also been found in the depths of the oceans in areas around thermal plumes.

Eubacteria include both aerobic and anaerobic forms. Two of the major groups are the Cyanobacteria and the Schizophyta. The Cyanobacteria are the “blue-green algae”. They are autotrophic and photosynthetic, but unlike plants, their chlorophyll is in the cytoplasm, not in discreet chloroplasts. Cyanobacteria contain chlorophyll a and a blue pigment, phycocyanin, which together give them their namesake color. They also contain other pigments, and when abundant, they can color the water in which they grow. The Red Sea and the Black Sea owe their names to “blooms” of Cyanobacteria.

Schizophyta are the largest groups of heterotrophic Eubacteria. Their classification is based on the Gram test, a staining method developed by a Danish doctor. Crystal violet stain is retained by the cell walls of certain bacteria. The cell walls of other bacteria lose the crystal violet complex by decolorization and are then counter stained. Bacteria that are stained appear purple under the microscope, and are termed Gram-positive. Those that do not take up the stain appear light pink and are termed Gram-negative.

Bacteria are also identified on the basis of their shape. Positive determination must be made by biochemical analysis. The three major bacterial forms are readily identified under the microscope. Spherical bacteria (cocci) may grow close together but do not link. Some species are causative agents of diseases such as strep throat, pneumonia, and gonorrhea. Rod-shaped bacteria (bacilli) includes species that cause anthrax and tetanus, while the spiral-shaped bacteria (spirilli) include species causing cholera and syphilis. However, very few of the large numbers of bacterial species are pathogenic, since the majority are beneficial members of Earth’s community, breaking down wastes, recycling nutrients, and enabling higher organisms to digest their food.

General Technique

Caution: Do not work with pathogenic (disease-causing) organisms unless you have had sufficient training and experience in their handling. Treat all bacteria as being pathogenic, both to establish proper work habits and because of the chance (however small) of a culture are inadvertently becoming contaminated by a pathogen. Even non-pathogens supplied by WARD’S are pure cultures and are heavily concentrated, and, as such, should be handled with care.

1. Sterilization
   1. Media and glassware: Use an autoclave or pressure cooker at 15 lbs. for 15 min. at 100°C.
   2. Glassware: Dry heat in oven at 160°C for at least two hours.
2. Wear a clean lab coat, smock, or apron to protect clothing and to reduce possible contamination of cultures.
3. While in the lab, avoid any hand to mouth operations, such as eating, smoking, or licking adhesive labels.
4. Wash hands thoroughly with soap and water, both before and after working with cultures.
5. Keep work surface clear of any unnecessary objects (e.g., books, purses, etc.)
6. Wash work surface with a capable disinfectant, such as 10% Lysol, 70% alcohol, or household bleach both before and after working with cultures.
7. Culture transfers - The only articles of equipment needed to make a transfer from the initial culture to a tube of sterile medium are a Bunsen burner or similar heat source, and an inoculating loop, needle, or sterile swab.
   1. Hold both tubes in the left hand (Fig. 1).
      
   2. With the needle in the right hand, pass the entire length of the wire through the flame until it has all been red hot (Fig. 2).
      
   3. While still holding the needle, QUICKLY remove the caps or plugs from the tubes, holding them between the fingers of the right hand (Fig. 3). Flame the mouths by passing them two or three times through the burner flames (Fig. 4). Hold these tubes almost parallel to the table top if they contain a broth. This will reduce the possibility of air-borne contaminants.
      
      
   4. Touch the needle to the medium in the culture tube to be sure it is cooled, and then to the culture mass. Apply the needle to the sterile medium in the other tube (Fig. 5). (This may be done either by streaking the surface of a slant, making a stab into a semi-solid media, or swirling the needle in a broth.) If Petri plates are used, place them on the table and lift the cover only enough to maneuver the needle when inoculating. It should be noted that it is not necessary to attempt to remove a large volume of the culture mass with the needle. A slight touch will place more than enough on the needle to make the inoculation.
      
   5. Flame the mouths of the tubes and cap them.
   6. Flame the inoculating needle until it is “red hot”.
8. If screw cap culture tubes are used, the cap should be kept loose to allow the aerobic cultures to get oxygen. (Most common bacteria and molds fall into this category.)
9. If cultures are to be kept for an extended period, however, they should be sealed tightly to prevent dehydration of the media. Then refrigerate to slow the metabolic processes of the organisms, unless the label states no refrigeration.

Procedures to follow in a biological spill:

Materials on hand at all times:

• Bio-hazard bag (autoclavable)
• Paper towels
• Gloves
• Tongs or similar instrument
• Disinfectant solution — 70% isopropyl alcohol, 10% Lysol, or household bleach in a squeeze bottle.

Procedure:

1. Pour disinfectant on all broken glass and contaminated surfaces. Extend coverage over 3” around original (contaminated) area.
2. Cover the spill area with paper towels. Add additional disinfectant to fully saturate them. Wait 30 minutes.
3. Wearing rubber gloves and using tongs, pick up all glass or residue along with paper towels and place in Bio-hazard bag.
4. Disinfect the area again by following steps 1–3.
5. Seal the Bio-hazard bag and autoclave the contents. Dispose.
6. Wash hands thoroughly.

Discarding of Microbiological Cultures

All cultures must be autoclaved prior to disposal.

1. Autoclave at 121°C, 15 psi for 15 minutes.
2. Contents of containers may be discarded. Note: Do not dump melted agar down the drain as it will later solidify and block flow.
3. Wash glassware in hot water and rinse well.

If an autoclave is unavailable, soak in bleach or incinerate.

Care of Cultures

When you receive your live cultures, they should be refrigerated to slow metabolic rates. The recommended medium and the optimum temperature for each bacterium is given on the tubes of the cultures supplied by WARD’S. The above technique is used for all but the following organisms:

1. Photobacterium fischeri
   1. P. fischeri, a marine bacterium, is one of the simplest light producing organisms and also one of the easiest to work with.
   2. Since P. fischeri will only produce light in fresh cultures, it is necessary to subculture it in order to observe its luminescent characteristics. This should be done 18–48 hours before the luminescence is to be observed.
   3. For best results, the room in which the observations are to occur should be completely darkened, and the eyes of the people who observe the experiments should be allowed to acclimate to the darkness. If this is not feasible, the room should be made as dark as possible and the experiments may be viewed in the bottom of a double paper bag with the opening of the bag held tightly about the viewer’s eyes.
2. Halobacterium salinarum requires a 25% salt medium for growth. It is a slow growing organism, taking 7–10 days at 37°C for agar slants. Twelve drops of Halobacterium solution (in 25% salt water) added to the tube and incubated horizontally in a slant rack will provide ample bacteria. Halobacterium can be stored at room temperature and can survive at temperatures up to 40°C.
3. Chromobacterium violaceum (pathogen) should not be refrigerated or subjected to cold temperatures as the culture will quickly die. Chromobacterium prefers to stay at 30°C and will keep for months this way.
4. Aquaspirillum prefers room temperature storage, although refrigeration will not hurt the culture. Spirillum volutans prefers to be held at 30°C. Rhodospirillum rubrum is photosynthetic and needs room temperature and a light source.

Lyophilized (freeze-dried) Bacteria Cultures

Under this mode of culture preservation, bacteria cultures will remain true-to-type for at least two years. It is recommended that these cultures be stored at 5–6°C, which is normal refrigeration temperature.

Culture re-establishment requires no sophisticated equipment or special technical experience. A serological pipet, along with general sterile technique, is all that is required. Please follow the instructions on the enclosed cards and be assured of success.

1. Using a sterile serological pipet, aseptically add to the lyophilized material no more than 0.5 ml of the appropriate sterile liquid transfer medium.
2. Mix well by drawing the hydrated cell suspension up and down through the pipet at least ten times.
3. Using a sterile swab, inoculate the agar.
4. Put remaining culture in broth tube. Re-established culture may then be sub-cultured onto growth medium.
5. All materials used (shell vial, pipet, etc.) should be autoclaved prior to disposal.
6. Incubate agar tubes horizontally and agar plates agar side up.

Given proper treatment and conditions, freeze-dried cultures will grow out in 24 – 48 hours. Some strains may exhibit a prolonged lag phase and should be given twice the normal incubation period before discarding as unviable.

Observing Bacteria under the Microscope

1. Spread a thin film from the culture on one half of a microscope slide, using the inoculating loop or needle.
2. Let the film dry.
3. Flame-fix the film by passing the smeared end of the slide through a Bunsen flame several times.
4. Flood the slide with 1% Methylene Blue stain (several drops will do).
5. Let stand for several minutes, then dip the slide into a container of water to remove the excess stain.
6. Blot dry. Do not rub, as this may remove the prepared culture from the slide. Add a drop of immersion oil and examine under high power (100X).

This procedure gives a quick look at bacteria and will easily demonstrate bacteria shape.

Gram Staining Procedure

1. Flame-fix a bacterial smear as described above.
2. Add several drops of Gram’s Crystal Violet stain.
3. Let stand for one minute, then rinse by dipping in water.
4. Add several drops of Gram’s Iodine Solution to “set” the stain.
5. Let stand one minute, then rinse by dipping in water.
6. Tilt the slide, then add 95% alcohol as a destain. Add drop by drop, letting the alcohol flow over the stained smear. Do this until there is no more color in the runoff. This step is to remove the color from gram-negative bacteria. The gram-positive organisms will retain the purple color.
7. Counterstain with Gram’s Safranin stain. Allow to stand for about 15 seconds, then rinse as before. Blot away excess moisture and allow to dry.
8. Add a drop of immersion oil and examine under high power (100x).

The cell walls of gram-negative bacteria are chemically different. They have a higher lipid content, which allows the alcohol destain to wash the stain out of the cell. The most accurate determinations using the Gram technique are done with newer cultures, preferably less than 24 hours old, although to view spores in Bacillus sp. or Clortridium sp. older cultures are necessary.

This guide is also available in PDF format on wardsci.com.

Related Products

• WARD’S Bacteria Lab Activities
• Microbiology Culture Media and Supplies
• Moneran Living Specimens (including bacteria cultures, cyanobacteria cultures, moneran sets, archabacteria cultures, and eubacteria cultures)

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