ENVE 301: Environmental Microbiology Laboratory Pure Culture Techniques
"The whole of science is nothing more than a refinement of everyday thinking." - Albert Einstein, Physics and Reality 
Upon completion of this laboratory you should:
1. Be able to define the following terms:
2. Be able to explain why it is important to use aseptic transfer and dilution/isolation streaking techniques.
3. Be able to explain how to get isolated colonies by using the dilution/isolation streaking technique and why it is necessary to get isolated colonies.
4. Be able to explain why these procedures are done:
a. flaming the loop
b. inverting inoculated plates
c. leaving the lids on petri dish at all times except when streaking or collecting a sample
5. Be able to explain why these procedures are not done:
a. placing a contaminated loop on the bench
b. placing a hot loop into a liquid culture
c. placing a sterile loop on the bench during streaking
6. Be able to recognize contamination on an agar plate.
7. Given an incubated streak plate, be able to determine if streaking problems exist and to describe what the problems are.
Bacteria are everywhere! On the bench tops, in water, soil and food, on your skin, in your ears, nose, throat, and intestinal tract(normal flora). The diversity of bacteria present in our environment and on and in our bodies is incredible. When trying to study bacteria from the environment, one quickly discovers that bacteria usually exist in mixed populations. It is only in very rare situations that bacteria occur as a single species. However,to be able to study the cultural, morphological, and physiological characteristics of an individual species, it is essential, that the organism be separated from the other species that are normally found in its habitat. In other words we must establish what is called a pure culture of the microorganism. A pure culture is defined as a population containing only a single species or strain of bacteria. Contamination means that more than one species is present in a culture that is supposed to be pure. Contamination does not imply that the contaminating organism is harmful, it simply means that the contaminating organism is unwanted in the culture that you are trying to isolate and study. Special techniques, commonly referred to as aseptic pure culture techniques, must be used to obtain a single isolated strain for study.
Before learning the techniques involved in manipulating bacterial cultures, it is first necessary to become acquainted with the tools of microbiology.
Inoculating loops or needles are used to transfer microorganisms. Traditionally, inoculating loops/needles were made from either nichrome or platinum wire. Wire loops and needles are sterilized between uses by flaming the loop (incineration of the microorganisms). Loops and needles must be flamed before and after each use. To flame a loop, hold the loop so that the wire part of the loop (not the handle) glows red hot. All of the loop should be flamed before sterilization is considered to be complete.
Recently, presterilized plastic loops/needles (Figure 1) have gained widespread popularity for many uses in the microbiology laboratory. One advantage of plastic loops/needles is that they do not have to be sterilized before use. The disadvantage of these loops is that they are single use disposable items
Figure 1: Inoculating Loops and Needles Source: http://www.antonides.nl/en/entogen.htm
Petri dishes (plates) (Figure 2) are covered dishes used to culture microorganisms. The sterile petri dish is filled with a solid medium (agar) and the bacterial culture is applied to the surface or embedded within the medium
Figure 2: Petri Plates Source: http://www.netpath.net/~billbsr/Page26.htm
Media (sg. medium) is a general term referring to the substance upon which a microorganism is grown. Ideally, microbiological media are designed to mimic the environment in which the organism naturally grows. Potato slices and the vitreous fluid of cow's eyes are two examples of early microbiological media. (Historical note: the term inoculate meaning "to implant microorganisms or infectious material into a culture medium" [The American Heritage Dictionary] is derived from the Latin root inoculare - in meaning in and oculus meaning eye, probably reflecting the early use of vitreous fluid and cow's eyes as a medium for the growth of microorganisms). Fundamentally, microbiological media currently are categorized as either chemically defined or complex. In a chemically defined medium, the exact amount of pure chemicals used to formulate the medium is known. A complex medium is composed of a mixture of proteins and extracts in which the exact amount of a particular amino acid, sugar, etc. is not known.
Chemically Defined and Complex Media
Chemically Defined: Beijerinckia Medium. Used for the cultivation of Beijerinckia species
Amount per liter of medium (grams)
Preparation: Add components to distilled/deionized water and bring volume to 1.0 L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min @ 15 psi pressure.
Complex: Soil Extract Medium. Used for the cultivation of Histoplasma capsulatum, Blastomyces dermatitidis, and Bacillus species.
Amount per Liter of medium (grams)
Preparation: Add 500.0 g of garden soil to 1.0 L of tap water. Autoclave for 3 hours at 15 psi/121oC. filter through Whatman No. 2 filter paper. Add remaining components to filtrate. Bring volume to 1.0 L with tap water. Gently heat and bring to boiling. distribute into tubes. Autoclave.
In addition to the distinction between chemically defined and complex, microbiological media may be further categorized as enrichment, selective, and differential. An enrichment medium contains some important growth factor (e.g. vitamin, amino acid, blood component, or carbon source) necessary for the growth of fastidious organisms. Selective media allow for the selection of particular microorganisms that may be present in a mixed culture. Selective media usually contain a component that enhances the growth of the desired organism or inhibits the growth of competing organisms. Differential media allow for the separation of organisms based on some observable change in the appearance of the media. Any single medium may be a combination of the above categories. For example, Mannatol Salts Agar (MSA) for the isolation and identification of Staphylococcus sp. is a complex, selective, and differential medium. MSA contains NaCl, mannitol (a simple sugar), pancreatic digest of soy bean meal, postassium phosphate and phenol red (a pH indicator). The presence of the pancreatic digest in the medium make it a complex medium since the exact composition of this material is not known. The relatively high concentration of saltin the medium is designed to inhibit many organisms and to select for salt tolerant organisms. Staphylococcus is commonly found on the human skin - a salty environment due to sweat. Finally, the inclusion of mannitol and phenol red makes MSA a differential medium. Staphylococci metabolize mannitol with the production of acid as a waste product. This acid lowers the pH of the agar in the immediate vicinity of the organism. Phenol red changes color from red to yellow when the pH falls. Thus mannitol fermenting organisms can be differentiated from other organisms because the area around colonies of mannitol fermenters changes color from red to yellow as the organisms grow.
Microbiological media may be prepared as either liquid (broth) or solid media. When a solid medium is prepared, the corresponding broth is solidified by the addition of agar to the broth. Agar is a polysaccharide found in the cell walls of some algae. It is generally inert, from a bacteriological point of view -- very few microorganisms degrade agar. In addition, its thermal properties (melts at 100oC and solidifies at approximately 45-50oC) make it an ideal solidifying agent for microbiological media.
Fanny Angelina Eilshemius was born in 1850 in New York to a wealthy Dutch immigrant family. As a young woman, she toured Europe. While in Europe, she met and married (1874) a German doctor, Walther Hesse. Dr. Hesse joined Robert Koch's laboratory in 1881 to study the new science of microbiology. Frau Hesse, nicknamed Lina, was a talented artist and drew illustrations for her husband's publications. At this time, gelatin was used as a solidifying agent for microbiological media. Gelatin plates frequently melted on hot days and many of the bacterial isolates digested (liquefied) the gelatin. According to Wolfgang Hesse (ASM News Vol 58#8 p. 425-428, 1992). a descendant of Dr. and Frau Hesse, Dr. Hesse asked his wife why her jellies and puddings stayed solid in warm weather when his microbiological media did not. She told him that she used agar-agar as a solidifying agent when she made jellies in hot weather. She had learned of agar-agar as a youngster in New York from a Dutch neighbor who had immigrated from Java. Dr. Hesse subsequently tried agar as a solidifying agent in bacteriological media and found that it worked wonderfully. Since then, agar has been the standard solidifying agent for microbiology.
Isolation of Pure Cultures From Mixed Cultures
Several different methods for getting a pure culture from a mixed culture are available. These include:
- Dilution to extinction
- Pour plate
- Streak plate
In this laboratory exercise, you will be learning the pour plate and streak plate techniques.
Both of these techniques depend upon the physical/spatial isolation of a single bacterial cell on/in a solid medium. In the pour plate technique, an inoculum is added to melted, cooled agar. The agar inoculum mixture is then poured into a petri dish. When it solidifies, isolated cells are trapped within the agar matrix. These cells give rise to isolated pure colonies of the bacteria. A colony is a visible mass of microorganisms growing on a solid medium. A colony is thought (in general) to have formed from reproduction of a single cell so that all the members of a colony are descendent from that original cell. In the streak plate technique, the inoculum is applied to the surface of an agar plate. By streaking the inoculum across the surface of the agar, a spatial separation of cells is achieved. For economy of materials and time, the streak plate method is best. It requires some skill to achieve a plate that reveals well-isolated colonies, however, good technique is forthcoming with experience and practice. A properly executed streak plate will give good isolation of colonies suitable for sub-culturing. The important thing is to produce good spacing between colonies.
When working with bacterial cultures, it is essential that you use proper aseptic culture techniques. Aseptic technique is the manipulation of microorganisms such that contamination by undesirable organisms is prevented. or the precautionary measures used to avoid contamination of cultures. Aseptic techniques not only protect a laboratory culture from becoming contaminated, but also protect the worker and the environment from becoming contaminated by the microorganisms. You should be able to perform standard aseptic techniques with ease and precision so that isolation and cultivation of bacteria in pure culture may be accomplished.
Procedure for Aseptic Transfer of Microorganisms (For additional illustrations of aseptic transfers try this site)
- Make sure you have all the cultures, media and necessary materials.
- Check the condition of the media, looking for such undesirable conditions as:
- contamination: evidenced by the presence of colonies of bacteria or fungus on a solid medium or a clear broth turning cloudy.
- solid media will pull away from sides of a container when the medium is too dry.
- Liquid media will have a lesser volume of broth.
- excessive moisture:
- appears as numerous droplets of water or a thin film of water on the surface of solid agar.
- the presence of excessive moisture increases the likelihood of contamination.
- Label all media to be used. Label should always include:
- Microbe or Sample Name/Incubation Temperature
- Turn on the Bunsen burner and light the gas Make sure that the flame is no more than four inches high and that the blue inner cone is no more than two inches high.
- Sterilize the inoculating loop or needle by heating it in the flame. Hold the instrument like a pencil and at an angle so that the wire is pointing downward. Pass the entire wire portion through the flame starting at the base, where it is attached to the holder, and continue all the way through the loop or end of the needle. Heat the wire until it is glowing red then allow it to cool before picking up any microorganisms. The instrument is now considered to be sterile. Do not touch the wire to any surface, or it will become contaminated and it will be necessary to repeat the sterilization procedure.
- Remove the inoculum
- From a broth culture (organisms growing in a liquid medium)
- Hold the culture tube in one hand and gently resuspend the organisms. DO NOT SHAKE THE TUBE UP AND DOWN AS THE CULTURE MAY SPILL
- In your other hand, hold the sterilized inoculating loop as if it were a pencil
- Remove the cap of the culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated. Very briefly flame the lip of the culture tube to create a convection current which forces air out of the tube. This prevents contaminants in the air from entering the tube.
- Keeping the tube at an angle, insert the loop and remove a loopful of material. Note the broth and associated microorganisms should stick to the loop due to surface tension forces.
- Again, flame the lip of the culture tube and replace the cap.
- From a solid medium
- Lift the lid of the petri plate. The less you can lift the lid, the less your chance of airborne contamination.
- Using your loop, touch the edge of a discrete colony. A small amount of material should attach to the loop.
- Transfer the inoculum to a sterile medium
- To a broth tube
- Pick up the sterile broth tube and remove the cap with the little finger of your loop hand. Again, do not set the cap down.
- Flame the lip of the tube, place the loopful of inoculum into the broth and withdraw the loop. Do not lay the loop down on the table top after this step.
- Flame the lip of the tube and replace the cap
- Resterilize the loop by flaming if it is a metal loop or dispose of the loop if it is a plastic loop in the infectious waste container on your desk
- To a solid medium
- Lift the edge of the petri dish lid just enough to insert the loop
- Streak the loop across the surface of the agar. Avoid digging into the agar by keeping the loop close to horizontal during the streaking.
- Remove the loop and close the lid
- Resterilize or dispose of the loop.
The pour plate technique, also sometimes called the loop dilution method, involves the successive transfer (serial dilution) of bacteria from the original culture to a series of tubes of liquified agar. Basically, a loopful of your original culture is transferred to a tube of liquifeid agar and mixed. As a result of this transfer, the concentration of bacteria in the first tube is lower than the concentration in the original culture; in effect you have diluted the original culture. A loopful of material from the first tube of liquified agar is then transferred to the second tube, effecting an additional dilution of the bacterial culture. The process is repeated for a third tube of liquified agar. Following inoculation of the tubes of liquified agar, the contents of each tube is poured into a separate Petri plate. After incubation, one of the plates should have an appropriate number of colonies to allow separation and isolation.
The pour plate technique is technically easier than the streak plate (surface dilution) technique; although, it certainly is not foolproof. In addition to all of the concerns regarding aseptic technique, a major concern in preparing pour plates is the temperature of the agar.
Tubed of liquified agar (called deeps or pours) are prepared by heating in a boiling water bath (or microwave) until melted (100oC). They are then cooled to 50oC in a water bath. When you remove the deeps from the water bath, they cool down. Agar will solidify at about 45oC. If it solidifies it must be boiled again to remelt it. If your agar deeps solidify in the tube before they are inoculated, it is time consuming and invonvenient. If they solidify after inoculation but before the agar is poured into petri plates, remelting the agar will kill the inoculated organisms. Under these circumstances, there is nothing to do but start over. The key to successful pour plates, then, is to be well organized and work quickly.
Supplies and Equipment Needed (per student)
- Mixed culture of bacteria: E. coli, Micrococcus luteus and Serratia marcescens
- Nutrient agar pours (3)
- Sterile petri plates (3)
- Inoculating loops
- Bunsen burner
- Marking pen
- Nutrient agar slants
In today's laboratory, you will be working with a mixture of three bacterial species - Escherichia coli (E. coli), Micrococcus luteus (M. luteus) and Serratia marcescens (S. marcescent). A brief description of these organisms follows.
E. coli is quite likely the most studied and best understood organism on the face of the globe. The standard organism of basic research laboratories in bacterial metabolism, physiology, and genetics, it is also widely used in genetic engineeringas a host organism for foreign DNA. Outside of the laboratorym the organism is an inhabitant of the intestinal tract of warm-blooded animals. The organism is excreted with fecal material. Because of this E. coli is widely used as an indicator organism for the presence of sewage contamination (see Lab 5).
E. coli is a gram negative rod shaped bacterium It forms creamy-white colonies on Nutrient agar when incubated at either 35oC or room temperature.
M. luteus is a gram positive, coccus shaped bacterium belonging the the family Micrococcaceae. Cells of M. luteus generally occur in tetrads (groups of four) or clusters and are not motile. The organism may be found on human skin where it functions as a commensal organism (it does not cause disease). The organisms produce carotenoid pigments and thus form yellow colonies on nutrient agar.The organism grows slowly at 35oC and hardly at all at room temperature.
S. marcescens is a gram negative rod shaped organism. It occurs naturally in soil and water as well as in the intestines. One of the unique characteristics of this organism is the production of a red pigment - prodigiosin - at room temperature, but not at elevated temperatures. You will use prodigiosin production as a way to identify S. marcescens on your streak plates and to differeentiate it from E. coli.
In addition to its taxonomic use, prodigiosin has had a profound effect on world history. In 1263, the Miracle of Bolsena occurred. The German priest, Peter of Prague, was breaking bread for communion at the Church of Saint Christina in Bolsena, Italy. When he broke the communion wafer, he saw it had blood on it - or at least it had a dark red blood-like substance. This was interpreted as an example of transsubstantiation of the host - the transformation of the communion wafer into Jesus's flesh. One year later, in 1264, Pope Urban institued the feast of Corpus Christi (Body of Christ) to commemorate this miracle. S. marcescens growing on bread produces copious amounts of prodigiosin and colonies of the organism therefore look remarkable like blood.
On the other hand, the discoloration of bread by the appearance of blood/prodigiosin has also had catastrophic results. Starting in the year 1155, with the story of William of Norwich, antisemites have used the appearance of blood on matzah (unleavened bread eaten at Passover) as support for the blood libel against the Jews and as a justification for massacres and the expulsion of jews from many areas.
Finally, in more recent history, S. marcescens was used as a surrogate organism for research on biological warfare. In the U.S. biological warfare program, the bulk of the research was based at Ft. Detrick and used "surrogate biological agents" to model more deadly organisms. Most of the offensive tests were based on "secret spraying" of organisms over populated areas. This program was shut down in 196 9. One of the biggest experiments involved the use of Serratia marcescens being sprayed over San Francisco. This organism is especially nice because it produces a red/pink pigment when grown on certain media, which makes identification very easy. A t one point, 5000 particles/minute were sprayed from the coastal areas inward. During this time, 1 man died(in the hospital) and 10 others became infected in what was described as "a mystery to doctors." Although the military never did many follow up stud ies on these tests, one results was that it showed nearly every single person became infected with the test organism. In hindsight, now that some of this information has become declassified, it's been shown that during periods following spraying tests, there were 5-10 times the normal infections reported. Other experiments included tests on Minneapolis that were disguised as "smoke screen tests" because residents were told a harmless smoke was being tested so that cities might be 'hidden' from radar guided missiles.
- Disinfect your table top
- Label the bottom surface of three sterile petri plates with you name and date. Label the bottom surface of the plates I,II, and III, respectively.
- Obtain three tubes of nutrient agar pours. Label the tubes I, II, and III respectively. Place the tubes in a beaker of hot tap water to prevent the agar from solidifying.
- Inoculate tube I with one loopful of the mixed culture. Gently mix and return the tube to the beaker of hot tap water.
- Inoculate tube II with one loopful of tube I. Gently mix and return the tubes to the beaker of hot tap water.
- Inoculate tube III with one loopful of tube II. Gently mix and return the tubes to the beaker of hot tap water.
- Sequentially pour the contents of each tube into the corresponding petri dish. Make sure to flame the lip of the tube when you open it.
- Allow the agar to solidify at room temperature.
- Incubate the plates in an inverted position for 24-48 hours at 35oC
- Examine your plates for isolated colonies. Record the appearance of the plate.
- Pick a small inoculum from an isolated colony of each type of organism in the mixture. Transfer it to an agar slant. Incubate for 24-48 hours and gram stain to check for purity. You must retain your pure cultures (slants) and gram stains for me to check.
Streak Plate Technique
The dilution/isolation streaking procedure, originally developed by Loeffler and Gaffky in Koch's laboratory, involves the dilution of bacteria by systematically streaking them over the agar surface in a petri dish to obtain isolated cells which will subsequently grow into piles of cells, or isolated colonies. If the agar surface grows microorganisms which are all the same genetically, the culture is considered a pure culture.
In the streaking procedure, a sterile loop or swab is used to obtain a microbial culture. The inoculating instrument is then streaked lightly over an agar surface. On the initial section of the streak, many microorganisms are deposited resulting in confluent growth,which is growth over the entire surface of the streaked area. However, because the loop is sterilized between streaking different sections, or zones, fewer and fewer microorganisms are deposited as the streaking progresses. Finally, only an occasional microorganism is deposited, because the streaking process dilutes out the sample that was placed in the initial section.
During incubation, the isolated microbes multiply, giving rise to individually isolated colonies in the lightest inoculated areas. Colonies appear as piles of material on the agar surface, and they come in a variety of shapes, sizes and textures which are characteristic of individual microorganisms. If a single Escherichia coli cell is deposited on a nutrient agar plate and incubated at 37°C, the cell and its progeny will divide every 30 to 40 minutes. In 10 to 12 hours, the colony will have reached a population of one million and a pinpoint colony will be visible.
To obtain good results with this technique, the agar surface should be smooth, moist, and free of contamination. However, excessive moisture from the condensation of water, derived from the initial cooling of the hot sterile media, can collect on the inside of the lid and sides of the plate. If this water drops down onto the agar surface, spreading and merging of colonies can occur. This is why one should invert the plates after streaking them and when they are incubated.
Supplies and materials Needed (per student)
- Mixed culture of bacteria
- Nutrient agar plates
- Inoculating loop
- Bunsen burner
- Marking pen.
- If you have not already done so, disnfect your work area.
- Label the bottom surface of the sterile petri plate with your name and the date.
- (Optional, but recommended) using the marking pen, draw lines to divide the plate into four quadrants. I will demonstrate the size and shape of the quadrants to be used.
- Obtain a loopful of the mixed culture. Be sure to use aseptic transfer technique.
- Streak the inoculum into the first quadrant as shown in the Figure below
Streak plate Quadrant 1 Source: http://www.cat.cc.md.us/~gkaiser/labmanua/lab3/fig2a1.html
- Flame the loop to sterilize it. DO NOT OBTAIN A NEW INOCULUM. Using the sterile loop streak from quadrant 1 to quadrant 2 as seen in the figure below. Be sure that you streak several times through quadrant 1 to pick up some organisms on the loop.
Streak Plate Quadrant 2
- Repeat the same general procedure for quadrants 3 and 4 respectively. Be sure to flame your loop between each quadrant.
- Flame the loop to disinfect it. Invert the plates and incubate for 24-48 hours at room temperature.
- During the next laboratory period examine your plates for isolated colonies, Record the appearance of the plate. A plate with well isolated colonies looks like this
Assignment: Upon completion of the laboratory you should be able to:
1. Describe the steps you would take in order to eventually obtain pure cultures of each organism.given a mixture of two bacteria and plates of Nutrient agar,
2. .Using the streak plate method of isolation, obtain isolated colonies from a mixture of microorganisms.
3.Pick off isolated colonies of microorganisms growing on a streak plate and aseptically transfer them to sterile media to obtain pure cultures.
4.Define: selective medium, differential medium, enrichment medium, and combination selective-differential medium.
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