Pure culture techniques

Pure culture techniques
1st sem MSc

1. ISOLATION OF PURE CULTURE TECHNIQUES

2. INTRODUCTION : CULTURE – Act of cultivating microorganisms or the microorganisms that are cultivated . The cultivation of microorganisms , bacteria , or of tissues , for scientific study medicinal use , etc . There are two types of cultures : Mixed culture Pure culture Mixed culture : Cultivating more than one microorganisms Pure culture : Containing single species of organism A pure culture is usually derived from a mixed culture(one containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample many times before inoculating the new medium . KKR1116 2

3. WHY PURE CULTURES ARE IMPORTANT IN MICROBIOLOGY ? Pure cultures are important in microbiology for the following reasons Once purified the isolated species can then be cultivated with the knowledge that only the desired microorganism is being grown . A pure culture can be correctly identified for accurate studying and testing , diagnosis in a clinical environment . Testing / experimenting with a pure culture ensures that the same results can be achieved regardless of how many times the test is repeated . KKR1116 3

4. THE ISOLATION TECHNIQUES ARE DILUTION SPREAD PLATE STREAK PLATE POUR PLATE MICRO MANIPULATOR METHOD KKR1116 4

5. Serial dilution, as the name suggests, is a series of sequential dilutions that are performed to convert a dense solution into a more usable concentration. In simple words, serial dilution is the process of stepwise dilution of a solution with an associated dilution factor. In biology, serial dilution is often associated with reducing the concentration of cells in a culture to simplify the operation. DILUTION KKR1116 5

6. Th objective of the serial dilution method is to estimate the concentration (number of organisms, bacteria, viruses, or colonies) of an unknown sample by enumeration of the number of colonies cultured from serial dilutions of the sample. In serial dilution, the density of cells is reduced in each step so that it is easier to calculate the concentration of the cells in the original solution by calculating the total dilution over the entire series. Serial dilutions are commonly performed to avoid having to pipette very small volumes (1-10 µl) to make a dilution of a solution. By diluting a sample in a controlled way, it is possible to obtain incubated culture plates with an easily countable number of colonies (around 30–100) and calculate the number of microbes present in the sample. OBJECTIVES OF SERIAL DILUTION KKR1116 6

7. Serial dilution involves the process of taking a sample and diluting it through a series of standard volumes of sterile diluent, which can either be distilled water or 0.9 % saline. Then, a small measured volume of each dilution is used to make a series of  pour  or  spread plates . Depending on the estimated concentration of cells/organisms in a sample, the extent of dilution is determined. For e.g., if a water sample is taken from an extremely polluted environment, the dilution factor is increased. In contrast, for a less contaminated sample, a low dilution factor might be sufficient. Serial two-fold and ten-fold dilutions are commonly used to titer antibodies or prepare diluted analytes in the laboratory. The dilution factor in a serial dilution can be determined either for an individual test tube or can be calculated as a total dilution factor in the entire series. SERIAL DILUTION FORMULA /CALCULATIONS : KKR1116 7

8. The dilution factor of each tube in a set: volume of sample volume of sample +volume of diluent For a ten fold dilution , 1 ml of sample is added to 9 ml of diluent . In this case , the dilution factor for that test tube will be : dilution factor = 1ml = 1 = 10 -1 1ml +9ml 10 KKR1116 8

9. Now, for total dilution factor, Total dilution factor for the second tube = dilution of first tube × dilution of the second tube. Example: For the first tube, dilution factor = 10 -1  (1 ml added to 9 ml) For the second tube, dilution factor = 10 -1  (1ml added to 9 ml) Total dilution factor = previous dilution × dilution of next tube total dilution of 10 -1  × 10 -1  = 10 -2 KKR1116 9

10. KKR1116 10

11. The following is the procedure for a ten-fold dilution of a sample to a dilution factor of 10 -6 : 1.The sample/culture is taken in a test tube and six test tubes, each with 9 ml of sterile diluents, which can either be distilled water or 0.9% saline, are taken. 2.A sterile pipette is taken. 3.1 ml of properly mixed sample/culture is drawn into the pipette. 4.The sample is then added to the first tube to make the total volume of 10 ml. This provides an initial dilution of 10 -1 . 5.The dilution is thoroughly mixed by emptying and filling the pipette several times. 6.The pipette tip is discarded, and a new pipette tip is attached to the pipette. 7.Now, 1 ml of mixture is taken from the 10 -1  dilution and is emptied into the second tube. The second tube now has a total dilution factor of 10 -2 . 8.The same process is then repeated for the remaining tube, taking 1 ml from the previous tube and adding it to the next 9 ml diluents. 9.As six tubes are used, the final dilution for the bacteria/cells will be 10 -6  (1 in 1,000,000). KKR1116 11

12. Serial dilution is performed in a number of experimental sciences like biochemistry, pharmacology, physics, and homeopathy. Serial dilution is used in microbiology to estimate the concentration or number of cells/organisms in a sample to obtain an incubated plate with an easily countable number of colonies. In biochemistry, serial dilution is used to obtain the desired concentration of reagents and chemicals from a higher concentration. In pharmaceutical laboratories, serial dilution is performed to receive the necessary concentration of chemicals and compounds as this method is more effective than individual dilutions. In homeopathy, homeopathic dilutions are used where a substance is diluted in distilled water or alcohol. It is believed than dilution increases the potency of the diluted substance by activating its vital energy. A PPLICATIONS KKR1116 12

13. Even though serial dilution is a useful technique in laboratories, it faces some challenges. Some of which are: An error might occur during the propagation of the sample, and the transfer inaccuracies lead to less accurate and less precise transfer. This results in the highest dilution to have the most inaccuracies and the least accuracy. Because serial dilution is performed in a stepwise manner, it requires a more extended period of time which limits the efficiency of the method. Serial dilution only allows the reduction of bacteria/cells but not the separation of bacteria/cells like in other techniques like flow cytometry. This technique also requires highly trained microbiologists and experts in aseptic techniques. LIMITATIONS KKR1116 13

14. Examples A simple example of serial dilution performed in our daily life is tea or coffee. In coffee, we add a certain amount of cold press coffee and add water over it so obtain a desired concentration of coffee. Another example of serial dilution is the dilution of acids and bases in chemistry to obtain a required concentration. Serial dilution of culture to determine the number of bacteria in a given sample through a plating technique is also an essential example of serial dilution. KKR1116 14

15. A variety of techniques has been developed for the isolation of microorganism, mainly the bacteria, from the specimen or from the sample cultures. The spread plate method is a technique to plate a liquid sample containing bacteria so that the bacteria are easy to count and isolate. A successful spread plate will have a countable number of isolated bacterial colonies evenly distributed on the plate. Spread plate culture technique is among the most widely used culture technique for isolating the bacteria. SPREAD PLATE TECHNIQUE KKR1116 15

16. KKR1116 16

17. KKR1116 17

18. In this technique, a serially diluted specimen containing 2 or more bacteria or microbe (Mixed culture) is used which is spread over the solidified agar media plates as a thin layer with the help of a sterile L-shape glass rod (Spreader) while the media plate is spun on a turntable. The principle behind this method is that when the Media plate is spun, at some stage, single cells will be deposited with the bent glass rod (Spreader) onto the surface of the Agar media. Some of the cells present in the specimen / diluted specimen will be separated from each other by a distance sufficient to allow the colonies that develop to be free from each other PRINCIPLE KKR1116 18

19. Make a dilution series from a sample. Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate. Dip the L-shaped glass spreader into alcohol. Flame the glass spreader (hockey stick) over a Bunsen burner. Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petri dish underneath at the same time. Incubate the plate at 37°C for 24 hours. Calculate the CFU value of the sample. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample. PROCEDURE KKR1116 19

20. The Spread Plate Technique, in conjunction with serial dilutions, is a valuable research tool. To demonstrate the cultural characteristics of the bacteria (e.g. color, texture, size, elevation, etc.). To isolate the bacteria in discrete colonies from the specimen containing more than 1 bacterium. For determining the Sensitivity and/or Resistance of bacterium towards the particular Drug/Antibiotics or Test substances. To obtain the sufficient  growth  of the bacterium for various biochemical and other tests. To estimate the viable counts of the bacteria in the specimen. To maintain the stock cultures. To transport or short-term storage of the specimen (e.g. stab culture). APPLICATIONS KKR1116 20

21. Strict aerobes are favored while microaerophilic tends to glow slower. Crowding of the colonies makes the enumeration difficult. Accurate dilutions using pipettes should be made. Volume no greater than 0.1 ml can be spread on the nutrient agar plate because it would not soak well and may result in colonies to coalesce as they form LIMITATIONS KKR1116 21

22. STREAK PLATE TECHNIQUE In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. The dilution or isolation by streaking method was first developed by Loeffler and Gaffky in  Koch’s  laboratory, which involves the dilution of bacteria by systematically streaking them over the exterior of the agar in a Petri dish to obtain isolated colonies which will then grow into the number of cells or isolated colonies. Streaking is rapid and ideally a simple process of isolation dilution. The technique is done by diluting a comparatively large concentration of bacteria to a smaller concentration. The decrease of bacteria should show that colonies are sufficiently spread apart to effect the separation of the different types of microbes. Streaking is done using a sterile tool, such as a cotton swab or commonly an inoculation loop. Aseptic techniques are used to maintain microbiological cultures and to prevent contamination of the growth medium. KKR1116 22

23. Limitations of Streak Plate Streak plating is a microbiology laboratory method that has two major disadvantages. Firstly, users will not be able to grow obligate anaerobes using this method. Secondly, only organisms that were viable in the original sample are able to be grown KKR1116 23

24. KKR1116 24

25. The streak plate method is a rapid qualitative isolation method. The techniques commonly used for isolation of discrete colonies initially require that the number of organisms in the inoculums be reduced. It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. The resulting diminution of the population size ensures that, following inoculation, individual cells will be sufficiently far apart on the surface of the agar medium to effect a separation of the different species present. In the streaking procedure, a sterile loop or swab is used to obtain an uncontaminated microbial culture. The process is called “picking colonies” when it is done from an agar plate with isolated colonies and is transferred to a new agar or gelatin plate using a sterile loop or needle. The inoculating loop or needle is then streaked over an agar surface. On the initial region of the streak, many microorganisms are deposited resulting in confluent growth or the growth of culture over the entire surface of the streaked area. The loop is sterilized by heating the loop in the blue flame of the Bunsen burner, between streaking different sections, or zones and thus lesser microorganisms are deposited as the streaking progresses. The streaking process will dilute out the sample that was placed in the initial region of the agar surface. PRINCIPLE KKR1116 25

26. KKR1116 26

27. There are many different types of methods used to streak a plate. There are two most commonly used streak patterns, a three sector “T streak” and four-quadrant streak methods. Picking a technique is a matter of individual preference and can also depend on how large the number of microbes the sample contains The streak plate technique is the most popular method for isolating specific bacteria from a sample containing a mixture of microorganisms. Streak plate technique is used to grow bacteria on a growth media surface so that individual bacterial colonies are isolated and sampled. Samples can then be taken from the resulting isolated colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested. When the bacteria are streaked and isolated, the causative agent of a bacterial disease can be identified. APPLICATIONS KKR1116 27

28. In nature, microbial populations do not segregate themselves by species but exist with a mixture of many other cell types. In the laboratory, these populations can be separated into pure cultures. These cultures contain only one type of organism and are suitable for the study of their cultural, morphological, and biochemical properties. At times also the determination of viable cells is very crucial in many microbiological procedures. To accomplish this, the serial dilution–agar plate technique is used. Briefly, this method involves serial dilution of a bacterial suspension in sterile water blanks, which serve as a diluent of known volume. Once diluted, the suspensions are placed on suitable nutrient media. The pour-plate technique is the procedure usually employed. POUR PLATE TECHNIQUE KKR1116 28

29. KKR1116 29

30. The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette. Molten agar cooled to 45°C, is poured into a Petri dish containing a specified amount of the diluted sample. Following the addition of the molten-then cooled agar, the cover is replaced, and the plates gently rotated in a circular motion to achieve uniform distribution of microorganisms. This procedure is repeated for all dilutions to be plated. Dilutions should be plated in duplicate for greater accuracy, incubated overnight, and counted on a Quebec colony counter either by hand or by an electronically modified version of this instrument. If the material to be tested is an aqueous liquid, a known volume is simply placed in the base of the Petri dish and 10–25 ml of molten culture medium (typically tryptone soy agar or plate count agar at 45o C) is poured onto it and quickly mixed by gentle swirling, then the plate is placed on one side for the agar to set. If the sample is a solid that is soluble in water that solid would normally be dissolved, but if it were insoluble then a suspension would be used. The problem in the last case would be to ensure that the suspension remained uniformly dispersed during pipetting and any dilution steps. Because the sample is dispersed through the medium before the gel sets, the colonies that grow are similarly dispersed throughout the agar. KKR1116 30

31. 1.Label around the edge of the bottom (not the lid) of a sterile but empty Petri dish with at least your name, the date, the type of growth medium, and the type of organism to be added to the melted agar medium. Include the dilution factor if plating serial dilutions, or a series of repeated dilutions, which results in a systematic reduction in the concentration of cells in the sample. Preparing serial dilutions is necessary if the number of cells in the sample exceeds the capacity of the agar plate, in which the statistically significant range is 30 to 300 CFU. If there are more than 300 CFU on a plate, then the colonies will be crowded and overlapping. 2.Obtain a tube containing 18 ml of melted agar medium. The agar medium should be dispensed into test tubes and pre-sterilized in an autoclave. On the same day, it is needed for an experiment, the agar should be melted in a steamer for 30 minutes then transferred to a 55 °C water bath. Only as much agar as is needed for the experiment should be melted as it cannot be re-used. Ten minutes before pouring plates, the tubes of melted agar should be transferred from the 55 °C water bath to a heat block on the laboratory bench set at 48 °C. Once the agar reaches this temperature, it is ready to pour. If the agar is too hot, the bacteria in the sample may be killed. If the agar is too cool, the medium may be lumpy once solidified. KKR1116 31

32. 3.Obtain your sample, which should be either a broth culture or a suspension of cells produced by mixing cells from a colony into buffer or saline. The samples may be derived from a dilution series of a single sample. The sample volume to be plated should be between 0.1 and 1.0 ml. 4.Open the lid of the empty Petri dish, and dispense your sample into the middle of the plate. Close the lid. Use aseptic technique throughout this procedure. Use either a serological pipette or micropipette to transfer your sample to the plate. Control the flow of the sample so it does not splash out of the plate. 5.Remove the cap from the tube of the melted agar, and pass the rim of the open tube through the flame of the Bunsen burner. 6.Open the lid of the Petri dish containing your sample and pour the agar in carefully. Close the lid then mix the sample with the agar by gently swirling the plate. 7.Allow the agar to thoroughly solidify before inverting the plate for incubation KKR1116 32

33. This technique is used to perform viable plate counts, in which the total number of colony-forming units within the agar and on the surface of the agar on a single plate is enumerated. Viable plate counts provide scientists a standardized means to generate growth curves, to calculate the concentration of cells in the tube from which the sample was plated, and to investigate the effect of various environments or growth conditions on bacterial cell survival or growth rate. APPLICATIONS KKR1116 33

34. The use of relatively hot agar carries the risk of killing some sensitive contaminants, so giving a low result. Small colonies may be overlooked. In the case of solid sample dissolving in water, some species may suffer a degree of viability loss if diluted quickly in cold water; consequently, isotonic buffer (phosphate-buffered saline for example) or peptone water are used as solvents or diluents. Colonies of different species within the agar appear similar — so it is difficult to detect contaminants. The reduced growth rate of obligate aerobes in the depth of the agar. Preparation for the pour plate method is time consuming compared with streak plate/and or spread plate technique. LIMITATIONS KKR1116 34

35. A  micromanipulator  is a device which is used to physically interact with a sample under a microscope, where a level of precision of movement is necessary that cannot be achieved by the unaided human hand. [1]  It may typically consist of an input joystick, a mechanism for reducing the range of movement and an output section with the means of holding a microtool to hold, inject, cut or otherwise manipulate the object as required. The mechanism for reducing the movement usually requires the movement to be free of  backlash . This is achieved by the use of  kinematic  constraints to allow each part of the mechanism to move only in one or more chosen  degrees of freedom , which achieves a high precision and repeatability of movement, usually at the expense of some absolute accuracy. MICROMANIPULATOR METHOD KKR1116 35

36.   Inverted microscope or stereo microscope with magnification up to 200 x, although 40—100 x should be sufficient in most cases. Phase contrast or dark field optics is an advantage Capillary tubes or haematocrit tubes — approx. 1 mm diameter x 100 mm long Bunsen burner or small flame Silicone tubing to fit over end of capillary tube. Length approximately 300—400 mm Hot plate with beaker containing distilled water Clean Glass slides Agar plates (1.5% Bacto -Agar, eg Difco Cat. No. 0140—01) made up in petri dishes (disposable, 90 mm diam.) Tissue culture plates Pasteur pipettes (sterile), rubber or silicon teat Sterile media, usually at dilute nutrient concentrations; e.g. f 20 , f 50 Sterile tissue culture multi-well plates or sterile disposable petri dishes (e.g. 33 or 55 mm diam or sterile culture tubes) EQUIPMENT KKR1116 36

37. KKR1116 37

38. 2.METHOD KKR1116 38

39. With a fine flame from a bunsen burner heat and draw out (holding at both ends) the capillary tube to form two micropipettes. The narrow end should be about twice the diameter of the cell to be micromanipulated. Heat distilled water to simmering point on hot plate. This is used for sterilizing the micropipette between each transfer. Place drops of sterile medium onto 1.5% agar plates with a sterile pasteur pipette. Alternatively place three drops on a glass slide. With silicone tubing attached to micropipette suck up and blow out with mouth a small amount of hot distilled water. This sterilizes the micropipette. Locate algal cell to be isolated in drop of enrichment sample. While observing the cell, suck up into the micropipette. Transfer the cell to a drop of sterile medium on agar plate or glass slide. Sterilize the micropipette. Repeat this process to “wash” the cell. The more times a cell is washed the less likely is bacterial contamination. However, the risk of cell damage increases with the number of times a cell is handled. The optimum number of washes will depend on the type of algae. Transfer the cell to dilute medium in a tissue culture plate, petri dish or culture tube. Place culture vessel under low light at appropriate constant temperature. Check microscopically for growth or wait until macroscopic growth can be detected (3—4 weeks after transfer). A clonal uni -algal culture should result from this method. KKR1116 39

40. For large non-motile cells or when time is at a premium an alternative method is to isolate into petridishes rather than on a slide. Multiple cells of a population are quickly transferred to the first rinse plate and then from there progressively fewer into subsequent plates. Because relatively less care is taken to avoid non-target cell transfer each petri dish acts as an enrichment culture with hopefully a relatively clean culture in the final plate (or even possibly clonal). The “clean” plates can then be used as the stock for making clonal isolations when time permits. KKR1116 40

41. Applications of micromanipulation techniques Micromanipulation in the food industry There is an increasing demand for thorough, low-cost quality control techniques that can certify a food item to be safe for consumption. In some cases, conventional methods of food safety are being replaced by micromanipulation techniques to test food products for gram-negative and gram-positive microbes. Transgenic animal production With the help of micromanipulation, several transgenic animals have been produced such as sheep, rabbits, and pigs. For instance, a transgenic mouse with a large body weight was produced by Palmiter   et al.  in 1982 by injecting the mouse embryo with a DNA fragment containing the rat growth hormones that were fused to the metallothionein I gene KKR1116 41

42. Electrophysiology Micromanipulators are being used extensively in combination with extracellular field recording in the study of epilepsy in rodents and vertebrate models such as zebrafish. Zebrafish have a genetic profile that exhibits a high degree of similarity with that of mammals, thereby making it easier to study gene mutation, knockdown, or genetic defects responsible for epileptic seizures. Genetics and gene therapy A study by Kizil  et al.  was carried out to understand how micromanipulation could be used in gene regulation. The study used the brain of an adult Zebra fish and introduced antisense morpholino oligonucleotides by cerebroventricular microinjection (CVMI) to knock down  in vivo  gene expression. Kizil  et al.  concluded that CVMI is an efficient technique for understanding adult neurogenesis and may help clinically in paving way for treatment of neurodegenerative disorders. At present, micromanipulation procedures are frequently used to carry out embryo biopsies to rule out any fetal anomalies due to a genetic disorder. Single-molecule micromanipulation has also proved to be of immense value in study of DNA and structural proteins enabling study of various parameters of individual proteins, such as binding and unbinding kinetics . KKR1116 42

43. In microbiology,  colonial morphology  refers to the visual appearance of  bacterial  or  fungal colonies  on an  agar plate . Examining colonial morphology is the first step in the identification of an unknown microbe. The systematic assessment of the colonies' appearance, focusing on aspects like size, shape, colour , opacity, and consistency, provides clues to the identity of the organism, allowing microbiologists to select appropriate tests to provide a definitive identification. COLONY CHARACTERSTICS KKR1116 43

44. KKR1116 44

45. KKR1116 45

46. KKR1116 46

47. KKR1116 47

48. KKR1116 48

49. Large opaque, round, creamy, white to yellowish colonies displaying beta-hemolysis on blood Staphylococcus aureus KKR1116 49

50. REFERENCE https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/Microbiology_Labs/Microbiology_Labs_I/08%3A_Bacterial_Colony_Morphology https://study.com/academy/lesson/serial-dilution-in-microbiology-calculation-method-technique.html https://microbenotes.com/serial-dilution/ KKR1116 50

51. THANK YOU KKR1116 51