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Laboratory 1

Objectives: Upon successful completion of this laboratory exercise you will be able to:

1. Streak agar plates for isolation of pure cultures;
2. Perform the Gram stain; and
3. Understand the role of selective and indicator media in the isolation and identification of bacteria.

4. Know how to identify Bacillus from cultural characteristics and Gram stain.

1. Observe the demonstration sheep blood agar plates (BAP), which have been streaked for isolated colonies with Escherichia coli, Staphylococcus aureus, viridans streptococci, Streptococcus pyogenes, Bacillus sp., and Neisseria sp.
2. Note the characteristics of colony color, morphology, and hemolysis of the isolated colonies on each demonstration BAP plate.
3. Observe the MacConkey (Mac) plate streaked with Salmonella typhimurium and E. coli.
4. Perform the Gram stain on liquid or plate cultures of each of the following organisms obtained from the demonstration plates: E. coli, S. aureus, S. pyogenes, Bacillus sp, and Neisseria sp.
5. Swab your tongue and use the inoculum to perform a Gram stain.
6. Gram stain and streak a broth culture, containing an unknown mixture of bacteria, for isolated colonies.
7. Record the all Gram stain results.

Observe the demonstration plates at each bench showing cultures which have been streaked for isolated colonies using the four-phase streak method described in the section "Colony Isolation" of Common Laboratory Techniques. Confluent growth is observed in the primary and secondary streaks, but in the third and fourth quadrants the streaking method has resulted in sufficient dilution such that isolated single colonies are present. Each isolated colony represents the clonal growth arising from a single cell that was present at that position on the plate.

Note the color and morphology of the well-isolated, single colonies on the demonstration plates. For a list of commonly noted colony characteristics, refer to Initial Evaluation of Bacterial Growth on Agar Surfaces, page xiii. Record the characteristic morphology of the organisms in the appropriate column of the table found at the end of this lab section.

Sheep blood agar plates are used for the inoculation of most clinical bacteriology specimens because this media supports all but the most fastidious clinically significant bacteria. The colony morphologies that commonly encountered bacteria exhibit on this medium also have been well documented. Certain bacteria produce extracellular enzymes that lyse (hemolyze) red blood cells in the surrounding agar. A wide, clear zone of complete hemolysis around colonies is called beta hemolysis, and is demonstrated around colonies of S. aureus. The narrower, greenish zones of hemolysis surrounding colonies of viridans streptococci are termed alpha-hemolysis. Alpha-hemolysis has unlysed red blood cells in an inner zone and an unidentified reductant of hemoglobin that causes the greenish discoloration. No discoloration or lysis of red blood cells is referred to as gamma hemolysis. Hemolysis can only be determined around well-isolated, single colonies and is accurately determined by holding a plate up to a light and observing the hemolysis with the light coming from behind. Hemolysis observed around areas of confluent growth may be due to nonspecific lysis of cells rather than to the action of hemolysin.

Before starting this work, review the list of General Rules and Safety Procedures. The Gram stain technique and information on flaming a loop are described in Common Laboratory Techniques on pages xiv and x, respectively.

A Difco Bactrol^TM Gram slide is provided for each student. It can be used to apply multiple specimens for gram staining. One square (labeled "C +/-") contains both gram positive and gram negative organisms. There are 9 other squares on the slide. Apply one sample per square of the following cultures:

from Demonstration 1 plates: Staphylococcus aureus, Streptococcus pyogenes, Neisseria sp., Bacillus sp., and Escherichia coli

from the metal rack in the center of each counter: tubes contain broth cultures of S. aureus, S. pyogenes, a mixture of TWO of today’s demonstration bacteria (labeled Demonstration Broth #1, 2, 3, 4, or 5), and a yeast.

The Demonstration 1 plates and the broth cultures of S. aureus, S. pyogenes, and the yeast are to be used by all students. Each student will stain only one of the Demonstration Broths. There will be a total of 10 specimens (including the control organisms in the C +/- square) to stain. Keep Demonstration Broth # 1, 2, 3, 4 or 5 in your rack for use later in the lab.

Note: A yeast culture has been added to show the size difference of these cells from that of bacterial cells. Before removing a sample from a broth culture, mix the culture either by rotation or by gently tapping the bottom of the tube. This will allow any microorganisms that may have settled to the bottom of the tube to become resuspended.

While the samples air dry, continue with Student Work 3. These samples will be Gram stained along with a specimen obtained in the following section.

Swab your tongue with the sterile swab provided. Rub the swab briskly over the back of your tongue. Firmly roll the swab over the surface of a clean glass slide. You may want to mark a circle on the back of the slide with a wax pencil to make it easier to tell where the sample is located.

Air dry and then heat fix this slide and the slide made previously (Student Work 2) and stain both at the same time.

After staining the slides above (Student Work 2 and Student Work 3), first microscopically view the "C +/- " section of the Difco Bactrol^TM Gram Slide. It should contain both gram-positive and gram-negative organisms. If it does not, the gram reactions of the other specimens on the slide may also be incorrect and new preparations should be made and gram stained (using a clean slide from your slide box). If both positive and negative bacteria are present in the "C +/- " section, observe the other sections of the slide. Note: When viewing the section of the slide containing the yeast cells, do not use the oil immersion lens—use the low and high power lens to view these cells.

Record the Gram reaction for each of the cultures and the cellular morphology (rods or cocci) and arrangements (chains, clusters, single cells, or diplococci) on the table at the end of this lab section. Draw representative cells or clusters of cells. Note if there is a difference in cellular arrangement of the bacteria between broth and agar cultures of the same organism. Verify proper results by having your Gram stains checked by your group instructor.

The tongue smear is a direct smear (a Gram stain from a clinical specimen). It may contain eukaryotic cells as well as normal bacterial flora. Record both the bacterial and eukaryotic cells observed. Be aware that the direct smear may contain bacteria that were not provided in today’s demonstrations.

MacConkey (Mac) agar is the most frequently used primary selective and differential agar. Gram-positive organisms are inhibited by crystal violet and bile present in this selective and differential plating medium. Gram–negative organisms that grow on MacConkey agar can be differentiated by the ability to ferment lactose.

Note that Gram-negative bacteria that are able to ferment lactose, such as E. coli, are bright pink on MacConkey (Mac) plates which contain the pH indicator neutral red owing to the production of acids, and they may be surrounded by a zone of precipitated bile. Non-lactose utilizers, such as Salmonella spp., utilize peptone in the plates and remain uncolored.

Likewise, there are selective agars used for the isolation of gram-positive organisms. One of those is Columbia Colistin-Nalidixic Acid (CNA) agar. CNA agar selectively inhibits the growth of gram-negative bacteria and is useful in the isolation of staphylococci and streptococci from clinical specimens containing mixed flora. NOT all gram-positive bacteria grow on these plates (most notably some Bacillus sp. are inhibited by CNA media).

STUDENT WORK 4: Streaking a culture to obtain isolated colonies.

Remove a sample from the Demonstration Broth #1, 2, 3, 4, or 5 that you gram stained in "Student Work 2: The Gram Stain" and streak the specimen for isolated colonies onto appropriate media (Remember to record the results from your Gram stain before streaking the plate). Media available for use are BAP, CNA, and Mac. See Appendix 2 on Media for more information about these agars. Note: If you do not see more than one type of Gram-stained organism in your culture, then do not use all three plates.

Each person must streak their culture to BAP plates. However from your Gram stain results, YOU must decide whether to inoculate a CNA plate or a Mac plate or both types of plates. For example, if your broth contains both gram-positive cocci and gram-negative organisms, you should use both a CNA and a Mac plate to allow you to initially isolate the gram-positive from the gram-negative organisms. If you have only gram-positive or gram-negative organisms, you only need to use one type of agar. Remember to streak for isolated colonies as described on pages xi-xii of the Common Laboratory Techniques section of this lab manual.

Label each plate appropriately (with name, Demonstration broth #, and date on the bottom of the plate—not on the lid) and place on a tray in your sector for incubation at 37⁰C. Plates should be held together by a rubber band (found in the center of each lab bench) and placed on the tray in an inverted position (agar side of petri dish on top).

 Bacillus spp.

General characteristics: Bacillus are aerobic, gram-positive, spore-forming rods.

As cultures age, they may stain gram-variable (both gram-positive and negative). Spores, which appear clear on Gram stain, may not be readily visible, as they do not stain with either crystal violet or safranin.

There are no standard methods for identifying Bacillus to the species level and no specific procedures for the direct detection of Bacillus in clinical material other than Gram stain of specimens. Bacillus is the only clinically relevant aerobic spore-forming organism. Therefore, the presence of a Gram-positive bacillus with spores on a stain of an aerobic culture confirms the genus identification.

Clinical significance: They are ubiquitous and unless isolated in large numbers, from sterile sites, or repeatedly from the same site they are most often considered culture contaminants. Two species: B. anthracis and B. cereus are most often associated with a disease process in humans.

B. anthracis, which is highly virulent, causes anthrax. Humans contract anthrax directly or indirectly from animals. Person-to-person transmission is exceedingly rare. There are three forms of anthrax infection: cutaneous, pulmonary, and gastrointestinal. Cutaneous cases account for 95-99% of the cases of human anthrax. These infections occur when spores are introduced through breaks in the skin or mucous membranes. Cutaneous anthrax is rarely life threatening and is readily treated. If recognized early, human pulmonary and gastrointestinal anthrax are also treatable. These forms are caused by either inhalation or ingestion of spores. Despite its publicity as a potential agent of biologic warfare, B. anthracis is not highly contagious and a large number of spores need to be ingested or inhaled to be lethal.

B. cereus is present in the soil and is the etiologic agent of two distinct food-poisoning syndromes: an emetic illness with a short incubation period (1-6 h), that resembles gastroenteritis due to S. aureus, and a diarrheal syndrome with a longer incubation period (6-24 h), that resembles food poisoning due to Clostridium perfringens. The heat-resistant endospores can survive inadequate cooking, and if the food is then held at a growth-permissive temperature for long enough, germination will occur, accompanied by production of enterotoxins. The two presentations are caused by two different enterotoxins. In this country, fried rice is the major food implicated in the emetic type of B. cereus food poisoning. The bacteria are commonly present on uncooked rice. The toxin that causes the emetic illness is heat-stable and will survive rapid cooking methods such as stir-frying. The diagnosis of B. cereus food poisoning can be confirmed by isolating > 10⁵ organisms per gram from the implicated food and recovering the same strain in significant numbers from acute-phase specimens (feces or vomitus) from the patient. In practice, this is an ideal situation that rarely occurs.

B. cereus is an opportunistic pathogen and can cause serious eye infection resulting from trauma. It has been isolated from wound infections, mostly in otherwise healthy persons, following surgery, traffic accidents, scalds, and burns.

Identification: It is important to be able to identify B. anthracis, even though it is rarely encountered. It can be suspected based on observation of colony morphology on BAP, microscopic morphology, and sensitivity to penicillin. The key characteristics for distinguishing B. cereus also include noting microscopic and colony morphology and performing selected biochemical tests. There are few clinical laboratories with personnel experienced in analyzing the colony and cellular morpohology of this genus and no standardized or commercial biochemical identification in routine use. Therefore, suspected isolates of B. anthracis are usually forwarded to a reference laboratory for confirmation.

Before you leave today:

• Review the Objectives for Lab 1 on page 1-1. If you need help understanding the proper technique for colony isolation, performing a Gram stain, or focusing your microscope, ask an instructor for help. It is expected that you will develop a competency in these skills within a few lab sessions.
• CLEAN YOUR LAB AREA WITH DISINFECTANT AFTER ALL LAB WORK IS COMPLETED
• WASH YOUR HANDS BEFORE LEAVING THE LABORATORY

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