M. Dalbey/Bio 105M Isolation of E. coli - Isolation of E. coli from an Environmental Sample
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1 Isolation of E. coli from an Environmental Sample We want to expand our horizons a bit beyond the domesticated lab strains of E. coli. In this exercise you will isolate "wild" E. coli strains from an environmental sample using several of the most widely used basic techniques in laboratory microbiology: Sterile Dilution Series Spread Plate/Colony Count Method Streak Plate Isolation The collection of E. coli strains will be further characterized in later exercises. Characterization of the strains in the culture collection will provide a glimpse into the genetic diversity of a natural population of E. coli. The source of strains will be a water sample from the Santa Cruz wastewater Treatment Plant. Specifically, the sample will be taken from the water flowing into the plant before any treatment is done. With regard to the meanness, or even the filthiness of particulars, for which (as Pliny observes) an apology is requisite, such subjects are no less worthy of admission into natural history than the most magnificent and costly; nor do they at all pollute natural history, for the sun enters alike the palace and the privy, and is not thereby polluted. Fancis Bacon, Novum Organum, 1620 AREN'T THERE GOING TO BE MANY TYPES OF BACTERIA PRESENT IN WASTEWATER? HOW WILL WE BE ABLE TO SPECIFICALLY ISOLATE E. coli? We must use selective growth conditions. We choose medium composition and incubation conditions that allow growth of the organisms we are interested in (E. coli); but prevent or inhibit the growth of other types of organisms present in the sample. Specifically, we will use a special growth medium called MacConkey medium and the cultures will be incubated aerobically at 37 C. WHAT FEATURES OF THE MEDIA AND INCUBATION ARE SELECTIVE FOR E. coli? Excellent question. The answer requires some familiarity with the basic properties of E.coli, relative to other organisms that may be present in the sample. Begin by reading the description of MacConkey medium posted on the class web site. Briefly, the crystal violet dye and bile salt detergent in the medium are toxic and inhibitory to a great many bacteria in nature. E. coli, and other bacteria from animal GI tracts tend to be resistant to these materials. The incubation temperature of 37ºC is too high for the growth of many bacteria normally inhabiting soil and water. E. coli, and other bacteria from the GI tract grow well at 37ºC. 1
2 Bacteria indigenous to the animal GI tract are bound to be a major component of the bacterial flora of the wastewater sample. Most of these bacteria are obligate anaerobes, meaning they cannot grow in the presence of atmospheric oxygen. E. coli is a facultative anaerobe, meaning that it can grow either in the presence or absence of oxygen. Finally, the medium contains the sugar lactose, which cannot be metabolized by most bacteria. E. coli metabolizes lactose to end products such as formic acid, lactic acid and acetic acid. When these acidic end products are excreted, the ph of the medium goes down. Acidification of the medium is indicated by a ph indicating dye and by bile salt precipitation, which forms a hazy halo around Lac + E. coli colonies. See images of bacterial colonies on MacConkey agar plates posted on the web site. 2
3 III. A. Procedure Day 1 Work individually, not in pairs. CAUTIONS The wastewater samples must be treated as potential biohazards. Do not pipette by mouth. Do not eat, drink or smoke in lab. Wear gloves, goggles, and your lab coat. Wash your hands and decontaminate the bench immediately after performing these steps. All contaminated materials should be placed in the proper containers for autoclaving before disposal. Do not pour fecal suspensions or bacterial suspensions down the drain. Do not put agar plates in the trash can. Clarify any uncertainty with the instructor before proceeding. Ethyl alcohol is flammable - keep it away from open flame. Use of the fire extinguisher will be demonstrated. 1. We will perform a serial dilution of the original wastewater sample for you. This will greatly reduce your potential exposure to biohazards. The wastewater is diluted in sterile 0.9% NaCl. Then we will give you a small tube with less than 1 ml total volume. Record in your notebook the dilution factor of the sample we give to you. 2. Using your P-200 micropipette, plate 0.10 ml (100 µl) from the diluted wastewater sample onto the surface of a MacConkey Agar plate. 3. Spread the cell suspension on the plate following the demonstration. Remember the Fire Hazard associated with this step. 4. Wait till the agar surface is DRY. (If the plates appear wet, they can be dried in the biosafety cabinet by exposing them to a laminar flow of sterile air.) Be sure the plates are properly labeled. Incubate them at 37 C, upside down. III. B. Procedure Day 2 1. Examine all the plates from Day 1 visually and without introducing contamination. You are interested in noting and describing the following: The number of colonies on each plate. How much variability is there in the size, morphology, color and texture of the colonies? Use a dissecting microscope to carefully observe these attributes. 2. Perform a streak plate isolation of one colony that appears to be Lac +. Streak onto another MacConkey plate. Be sure that you have provided a thorough description of the appearance of this particular colony in your notebook. See the graphic illustration below for more information on the streak plate isolation method. 3
4 3. Be sure your streak plate is properly labeled (name, date) and then place it in a 37 C incubator. This is an appropriate time to give your E. coli strain an official designation. This takes the form XY-1, where X and Y are letters of the alphabet. You can use your initials if you want. 4. After you have observed the plates and re-streaked a selected colony, you may use the semiautomated colony counters to count colonies. These plates were made from a natural sample with a diverse flora. Furthermore, no strategy of selective culture is 100% discriminatory. Therefore, we are faced with the uncertainty of deciding how large a colony must be before we accept it for counting. This judgment will be made collectively and uniformly in class. Use the results of the colony counts from the entire class to estimate the total number of bacteria and the number of E. coli per ml of wastewater. Report values in "colony forming units" (cfu) per ml, to 2 significant figures, in scientific notation. 4
5 IV. ASSIGNMENT 1. Write a one or two page summary of your observations of the Day 1 agar plates from the entire class. It must include descriptions of colony morphologies. 2. Calculation of total bacterial cfu/ml and the E. coli cfu/ml in the original wastewater sample. 5
6 STREAK PLATE ISOLATION 6
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