An experienced microbiologist employs many techniques and skills when handling microorganisms. More so than in any other field of science, the skills of a microbiologist need to be practiced and refined to achieve a level of proficiency. Microscopy is one skill you are already learning and will soon master with further practice. Other skills of the microbiologist allow for the safe handling and manipulation of microorganisms. Isolation and investigation of microorganisms are formidable tasks confronting both novice and experienced microbiologists. The skills employed to achieve these tasks are called aseptic technique.
Aseptic technique reduced the potential spread of bacteria used in laboratory to you or other people. This could happen if a pathogen were allowed to escape the lab on contaminated clothing, books, or other materials. You will be introduced this week to the basic skills of aseptic technique, and will be expected to apply and refine them over the course of the semester.
Aseptic technique is necessary to prevent contamination of pure cultures (a culture containing a single species) which are used routinely for analysis. Extraordinary precautions are needed to prevent contamination because microorganisms, particularly bacteria, are ‘everywhere’. Trillions of bacteria occur in, on and around our bodies, and even slight carelessness can lead to inadvertent contamination. The vast majority of these bacteria are harmless, even beneficial, but analysis of a contaminated culture will yield unpredictable and most likely erroneous results.
In Exercise III of this lab exercise, you will isolate an unknown bacterium, separate it from contaminants, and grow it as a pure culture. This will be the “semester unknown” that your team will maintain and analyze throughout the semester. Remarkably, when bacteria are collected from a site (such as by swabbing a surface) and transferred to standard bacterial culture media, only a small fraction of the cells present will actually grow. Those that do grow are abundant enough, but most species have specialized growth requirements that are not met by standard culture media.
Summary of exercise
I. You will practice making aseptic transfers between culture media.
II. You will practice making a streak plate using a provided mixed bacteria culture
III. You will inoculate a plate of nutrient medium from an environmental site to demonstrate the prevalence of microorganisms in the environment and use streak plating to isolate an unknown bacterial species.
IV. You will examine bacteria cultured on solid media to study the importance of colony morphology in identifying bacterial types.
I. Practicing Aseptic Transfers
The technique that you will use most frequently during the semester is aseptic transfer of bacteria, which you will do whenever you inoculate culture media with an organism. The inoculating loop (shown to the right) is the standard tool of the microbiologist and is used to transfer cells to and from culture media. An important point to remember is that seeing a mass of bacteria on the loop during a transfer is not necessary (although it is reassuring))even a single cell is adequate to start a new culture. An essential rule for any aseptic transfer is to sterilize the inoculating loop BEFORE and AFTER each transfer. Why? The techniques for doing aseptic transfers will be demonstrated in class. The manner in which you hold the tubes, caps and inoculating loop are important. You are encouraged to ask for assistance or a critique while you perform the transfers.
When fungal spores or bacteria-laden microscopic particles make contact with your plates, broths, and tubes colonies happily reproduce and your precious media eventually resemble something out of an abandoned full refrigerator. One can’t recognize individual colonies when the plates are covered with fuzz! No untreated surface in the lab is sterile, and nearly all dust and other particles have spores or active cells on their surfaces. Obviously, then, all labware and all media must be sterilized before use. Sterilization can be accomplished in several ways including the use of moist heat, filtration, ethylene oxide, radiation, or ultraviolet light. Moist heat will be employed for most sterilization procedures in the laboratory. Other methods are necessary for materials that won’t stand up to heat, such as untempered glass, some plastics, or labile chemicals such as most antibiotics.
A steam autoclave is an instrument that is designed to deliver steam into a chamber, generating high heat and pressure at the same time. Heating media to above 120 degrees C for 4 to 20 min. destroys nearly all living cells and spores. High pressure (typically 20 lbs/sq. in) allows the temperature to exceed 100 degrees, which can’t be accomplished with steam at one atmosphere. We use an autoclave that starts timing when the temperature reaches 121 degrees, and exhausts the steam slowly after the prescribed time above 121 degrees (to prevent exploding bottles!). The autoclave is effectively a giant pressure cooker.
To properly use an autoclave
Know the instrument – some are fully automatic, some are fully manual
Prepare supplies properly – the more layers or greater the volume, the longer it will take for the interior to heat up
Check the steam pressure and ensure that the instrument is set for slow exhaust if liquids are to be sterilized
Ensure that the door is closed properly and securely
Check that the time and/or automatic cycle are set properly
Ensure that the temperature is well below 100 degrees before attempting to open the door
Crack the door to allow steam to vent, keeping face and hands well away from the opening
Handle freshly autoclaved bottles and tube with care – use protective clothing and gloves. Materials may be superheated and explode or boil over on you.
Tryptic soy agar consists of a pancreatic digest of casein (milk sugar) and a papaic digest of soybean meal, with sodium chloride and agar. It is a general purpose medium for the culture of fastidious and nonfastidious microorganisms. Whatever we give you will grow on tryptic soy agar provided that you inoculate the plate with living material and culture it at an appropriate temperature.
Media are purchased as dehydrated granules or powder, and are rehydrated by mixing a measured amount of medium per measured volume of deionized water. Instructions for rehydration are usually printed on the container (40 gms/liter for tryptic soy agar). Since dry media tend to form clumps when dumped in water, it is best to layer the material on the surface and let it soak in. Containers used for media must have vented tops and should be capable of holding more than the intended volume of medium, to allow for expansion during sterilization. Agar does not distribute uniformly when melted. A safe way to ensure a uniform distribution for pouring plates or tubes is to drop a magnetic stir bar in the flask or bottle, then gently stir the medium after sterilization, while it cools. Stirring distributes the agar evenly. If screw cap bottles are used, the cap must be loosened prior to sterilization.
***CAUTION*** Exposing tightly stoppered bottles to variable pressures invites explosion and injury. When heating any liquids using any method, take care disturbing the flask or bottle. Material near the bottom may be superheated and boil over when moved. Stoppers, caps, covers, must be vented – never make them fit tightly.
Agar media for plates will be sterilized for 15 minutes in an autoclave in slow exhaust mode, stirred and cooled to about 50 degrees C, and plates prepared in the laminar flow hood. We use 1 liter bottles for plate media, with 500 ml medium per bottle. Sterile 100 mm plastic dishes come in sleeves of 20. To pour plates several stacks of new plates are placed in the hood and a minimum of 20 ml poured into each plate using aseptic technique. The hood prevents fungal contamination of the rich medium, which nearly always happens when we pour plates out on the lab benches.
Dishes can be left in the hood in a single layer with lids ajar or stacked with only the top lid ajar. Either way, we often get condensation which can lead to contamination of plates. To reduce condensation we may choose to leave fresh dishes for a period of time in the hood. One can expect to get 20 to 25 plates per bottle.
The only difference between broth and agar media is that broths are liquid and agars provide a solid support for growth. Use broth for faster growth, to get a uniform inoculum for dilution streaking, or for assays. In broth a species may display a characteristic pattern of association among individual cells, such as chains or clusters, that is not as obvious in agar cultures. Your unknown cultures will be prepared in broth, then mixed to give proportional amounts of each species prior to passing them out. Dry medium is layered onto the surface of a measured volume of water as with agar media, mixed, and distributed into individual capped tubes in racks. Racks are autoclaved and then allowed to cool, and caps tightened to prevent evaporation.
An agar slant consists of agar based medium in a culture tube. It is called a slant because the tube is placed at an angle during cooling to give a large slanted surface for inoculation. The tube can be tightly capped for relatively long term storage of an isolate with low risk of contamination or drying out of the culture. To prepare an agar slant, agar media are rehydrated just as they are for agar plates, however the agar should then be melted in a microwave oven, stirred to distribute it evenly, and distributed into capped tubes. The tubes should be filled just sufficiently to allow the agar to flow to just below the neck when the neck is laid over a horizontal 10 ml glass pipet. The tubes are autoclaved with caps loose as with all media, then laid on their sides using a pipet to keep them tilted up just enough to create a long slanted surface. After cooling, the caps are tightened and the tubes are ready for use.
The media on which you culture desirable microorganisms will also grow undesirable contaminants, especially molds and other types of fungus, and bacteria from your skin and hair. It is therefore essential that you protect your cultures from contamination from airborne spores and living microorganisms, surface contaminants that may be on your instruments, and from skin contact.
Never leave a culture dish open, even for a short time when viewing colonies of organisms, unless you intend to destroy it.
When it is necessary to open a dish, keep the lid close to the dish, open it only as far and as long as is necessary to accomplish the procedure, and keep the lid between your face (and your germs!) and the agar surface.
For most bacterial cultures you will use a sterile loop or needle to inoculate or to obtain an inoculum.
Flame a loop or needle to red-hot just prior to use, burning off any organic material
Cool the instrument by touching the sterile agar or liquid surface prior to touching a culture (or else you will kill it)
Re-sterilize the instrument after performing the procedure, putting down safely without burning the bench, you, or another student.
Pass the neck of a culture tube or any container with a culture or sterile contents through a flame before taking off the cap. Hold the cap with opening down, and the tube horizontal or nearly so. Convection from the heated neck will prevent dust from falling into the opening. Flame again before putting the cap back [see ‘preparing a bacterial smear’ in the staining section]
Use sterile disposable pipets to remove samples from a broth culture that must be kept uncontaminated.
Always be aware of where your hands are, where your face is, and whether or not your culture is in a position to be contaminated. If you have long hair, make sure it does not hang into your plate. Hair is full of potential contaminants, and is one of the principle sources of contaminating microorganisms.
If you have an open flame, long hair that is not tied back or loose clothing can be hazardous to your health.
A contaminated culture can often be rescued, however there is always the risk that you will re-isolate the wrong microorganism. Besides, you don’t have that kind of time to waste. Exercise extreme care to keep your cultures pure. You will have ample opportunity to practice media preparation, assay techniques, staining and observation, and especially proper culture and handling of microorganisms
To verify presence of bacteria in environment To be able to perform basic bacteriological transfers To learn aseptic technique To become familiar with characteristics of bacterial broth and agar slant cultures To understand what the pure culture is To practice spread plating, pour plating and streak plating techniques To be able to calculate cfu/ml and learn meaning of it To be able to distinguish basic features of bacterial colonies, broth cultures and agar slant growths To be able to recognize the advantages and —streak plate— limitations of culture characteristics in the identification of bacterial species
Procedure: Simplified and Combined
A) Microorganisms in the environment: Take sterile Nutrient agar plates
Test 1: Expose the surface of agar plate to air for
0 min à Group #1 5 min à Group #2 10 min à Group #3 15 min à Group #4 20 min à Group #5
Test 2: Expose the surface of agar plate to followings;
a) Touch the surface of the agar with your fingerà Group #6 b) Combine your hair over agarà Group #7 c) Cough onto agar 1 or 2 timesà Group #8
B) Transfer Broth Escherichia coli culture to sterile NB C) Use agar slant as a source of inoculum, transfer Bacillus thuringrensis to sterile NB with loop D) Inoculate a sterile agar slant by using Serratia marcescens broth culture E) Transfer to an agar deep from Escherichia coli broth culture to sterile deep agar with needle F) For colony selection use Bacillus subtilis agar plate to inoculate sterile NB *(always study aseptically; disinfect bench surfaces, work close to flame and put the loops and needs etc over flame to remove any potential contamination)
1. Serial Dilutions: Make serial dilutions by using saline solution (0,85 %NaCl) up to 10-5 , 10-6 and 10-7 as described below:
And use following chart for each group; Dilution Group # ——————– 10-5 1, 2, 3 10-6 4, 5, 6 10-7 7, 8
2. As a stock solution, use S. morcencens, while making solutions; be careful about mixing of solution. 3. After serial dilutions spread 0.1 ml of solution to plate and label together with dilution factor. 4. For pour plate, prepare hot and sterile NA and after cooling of agar mix 0.1ml of culture solution and pour directly to the plate. 5. Pour 0,1ml of diluted sample (in our group10-7 dilution rate) to plate by using spreader or rod. Firstly, flame spreader together with alcohol and cool it on the inner surface of Petri plate cover part, then spread sample gently and evenly. 6. To obtain discrete colonies from any kind of media such as NB or NA, take sample of bacteria by inoculum loop and by using “four corner method”. Firstly, sterilize loop over flame & cool it by waiting 10 sec, take sample from mixed culture tube and make parallel movements over agar, after that flame and sterilize loop again and take one line from this parallel lines by making one perpendicular line and make new & unrelated parallel lines and continue like this for two more time. *(do not forget to label plates correctly, write name of group, date, dilution, species name, spread volume and kind of plating technique whether spread or pour)
FINALLY INCUBATE ALL INOCULATED CULTURES, PLATES AND TRANSFERS AT 370C FOR 24 HOURS AND RECORD YOUR FINDINGS.
Results: A. Microorganisms in the Environment: Time of exposure to air #of colonies grown 0’ 0 5’ 5 10’ 9 15’ 15 20’ 13 Source of Bacteria #of colonies grown Touch the surface of the agar with your finger 37 Combine your hair over agar 14 Cough onto agar 1 or 2 times 29
B. Broth Transfer: Some colony formation was obvious when we look at cloudy appearance. C. Agar Slants as a Source of Inoculum: Some colony formation was obvious when we look at cloudy appearance. D. Inoculation of an Agar Slant: There are many colonies formed in the zigzag form on the surface. E. Transfer to Agar Deeps: Inside of the hole which is opened by needle contained many cells near the surface. F. Colony Selection: Some colony formation was obvious when we look at cloudy appearance.
(Colony numbers over 300 should be considered as uncountable)
Cfu/ml: #of colonies on plate x (1/dilution of solution)x(1/volume of inoculation) Acceptable results: 10-5à972 (1) cfu/ml=972*(1/10-5)*(1/0,1ml)=9,72*108 10-6à114 (2) cfu/ml=114*(1/10-6)*(1/0,1ml)=1,14*109 10-7à16 (3) cfu/ml=16 *(1/10-7)*(1/0,1ml)= 1,6*109
cfu/ml Ratios: (1)/(2)= 0,85 (1 is expected) (2)/(3)= 0,71 (1 is expected) (1)/(3)= 0,61 (1 is expected)
Colony # Ratios: (1)/(2)= 8,5 (10 is expected) (2)/(3)= 7,1 (10 is expected) (1)/(3)= 60,8 (100 is expected)
Discussion: In this experiment, we have learned several basic molecular biology techniques and use of equipments such as plating techniques (spread, streak, and pour).
Organisms live everywhere, especially microorganisms found in everyplace. In our experiments we showed that they are found in air and if you contact your nutrient media with air more and more you will get more number of colonies in general if conditions wind and place of nutrient media is similar or same. This is shown by up to 15 min, there were an increase in the number of colony but at 20 min there is a decrease in number. I think this is due to the place of plate remained opened and due to less circulation of air around open nutrient media. Moreover, I also can say simple although just one experiment is not enough finger touching can cause formation of more bacteria then coughing over it and combining of hair gives least number of colony, it means that our friend has clear hairs than any fingers and coughing. In addition to that it is necessary to note that it is normal to see bacteria colony formation from finger, hair because they are always contact with environment. Because Nutrient Broths have all necessary nutrients necessary for grooving of any kind of bacteria in general, we observed growing of colonies in all NB tubes. Although we expected that in the “deep agar” tube bacteria are grown at the bottom, whereas in the “slant” tube bacteria are grown on the surface. For agar deeps, as indicated in the result part we could see bacterial formation around and inside of the hole formed by needle. Moreover, that is because S.aereus is an aerobic bacterium and tends to go to the surface of the agar and there was red pigmentation specific to S.aereus. In the case of inoculation of agar slats by zigzag form, we observed zigzag formation of colony growth after 24 hours at 370C incubation. What about the serial dilutions and results? They are not successful in general, we could use just 3 results to calculate cfu/ml values. Moreover, results are not satisfying ten times decrease of colony number in the case of ten times dilution, in other words, ten times more colony formation in the previous dilution when we consider 16 colonies of 10-7 and 114 colonies of 10-6; ratio is 8,5. Reasons are, I suggest, improper dilutions, burst of cells in the case of strong wortexing although it is very difficult to kill bacterial cells by broken their cell wall. Actually, these unexpected results are expected if you are a new scientist working on a new subject. It is always possible to make errors; you should get experience by time with your failures. In our group and in many groups we could not obverse any colony formation in the pour plates but in some groups we could observe characteristic colonies which is red colored colonies of. During the dilutions we should use an isotonic solution like 0.85% saline solution to keep cells alive by preventing bursting due to osmolarity. One biologist or geneticist may be I should also add microbiologist should know the importance of leaving of Petri plates inverted after spreading on to agar plates in to the incubator. If they are not left inverted, the water vaporizes from the agar and condenses on the lid and falls back. That causes increase of the dilution of bacteria on agar and also loss of water from agar so form more stringent conditions to live in. We made three different types of plating techniwues; spread, streak, and pour. Spread plate was a general approach to study pure culture, isolating of pure culture. On the othe hand, streak plate was good to obtaining single colonies from any kind of stock like frozen or colony formed before. Pour plate was a good method to study bacteria which can live in an oxygen deficient environment and it was also easy to pour on just Petri plates together with proper mixing of desired bacteria. In our experiments, unfortunately, we failed to see colony formations neither on surface nor inside of NA. This may be due to killing of bacteria during addition to NA media, media might be very hot. As a final discussion, there were small colony formations and pink colored big colony formations in NA because we used mix culture for this purpose.
I think no need to mention aseptic techniques that should be follow by researcher. I can not imagine plating without studying close to fire or inside of laminar flow to prevent contamination and use of loop without flame sterilization until giving red color.
In conclusion, these exercises were successful and they were a good start to study and learn more basic microbiology methods, because they are essential of microbiology studies. New procedures were practiced, and further understanding of transfers, plating techniques, logic of serial dilutions and cfu/ml calculations were gained.
“Çalışmadan, yorulmadan ve üretmeden, rahat yaşamak isteyen toplumlar; evvela haysiyetlerini, sonra hürriyetlerini daha sonra da istiklal ve istikballerini kaybetmeye mahkumdurlar.” Mustafa Kemal ATATÜRK