micro lab ch 6.pdf

EXERCISE Standard Plate Count (Viable Count) 6-1 Theo...

EXERCISE Standard Plate Count (Viable Count) 6-1 Theory The standard plate count is a procedure that allows As shown in Figure 6.1, a serial dilution is simply a microbiologists to estimate the population density in a series of controlled transfers down a line of dilution blanks liquid sample by plating a very dilute portion of that (tubes containing a known volume of sterile diluent- sample and counting the number of colonies it produces. water, saline, or buffer). The series begins with a sample The inoculum that is transferred to the plate contains a containing an unknown concentration (density) of cells known proportion of the original sample because it is and ends with a very dilute mixture containing only a the product of a serial dilution. few-or no-cells. Each dilution blank in the series 0.1 ml 0.1 ml 1.0ml 1.0 ml 1.0 ml Original Tube 1 Tube 2 Tube3 Tube4 Tubes sample 9.9ml 9.9 ml 9.0ml 9.0ml 9.0ml diluent diluent diluent diluent diluent 10 - 2 dilution 1o- dilution 10 - 5 dilution 10 -s dilution 10 - 7 dilution ~-==== Plate A Plate D 10 - 5 ml original sample volume 10 -a ml original sample volume Plate B Plate C 1o-s ml original sample volume 10 - 7 ml original sample volume 6.1 Serial Dilution Procedural Diagram +This is an illustration of the dilution scheme outlined in the Procedure. The dilution assigned to each tube (written below the tube) represents the proportion of original sample inside that tube. For example, if the dilution is 10-4, the proportion of original sample inside the tube would be 1/1 O,OOOth of the total volume inside. When 0.1 ml of that solution is transferred to a plate, the volume of sample in the plate is 0.1 ml x 10-4 = 10-5 ml. receives a known volume from the mixture in the previous dilutions. Both types of dilutions can be calculated using tube and delivers a known volume to the next, typically the following formula, reducing the cell density to 1/10 or 1/100 at each step. V 1D 1 = V 2D 2 (Greater dilutions in single steps are generally avoided, because they can be accomplished more conveniently where V 1 and D 1 are the volume and dilution of the and with greater accuracy by simply combining 1/10 concentrated broth, respectively, while V2 and D 2 are the or 1/100 dilutions.) volume and dilution of the completed dilution. Undiluted For example, if the original sample in Figure 6.1 samples are always expressed as 1. Therefore, to calculate contains 1,000,000 cells/mL, following the first transfer the dilution of a 1 ml sample transferred to 9 ml of di- the 1/100 dilution in dilution tube 1 would contain luent, the permuted formula would be used as follows. 10,000 cells/ml (1,000,000 X 1/100 = 10,000 cells/ V 1D 1 1.0 ml X 1 1 ml). In the second dilution (tube 2) the 1/100 dilution D2 = - - - - = 10-1 V2 10 ml 10 would reduce it further to 100 cells/ml (10,000 X 1/100 = 100 cells/ml). Because the cell density of the As mentioned above, compound dilutions are calcu- original sample is not known at this time, only the dilu- lated using the same formula. However, because D 1 in tions (without ml units) are recorded on the dilution compound dilutions no longer represents undiluted sam- tubes. By convention, dilutions are expressed in scientific ple, but rather a fraction of the original density, it must notation. Therefore, a 1/10 dilution is written as 10-1 be represented as something less than 1 (e.g., 10-1, 10-2 , and a 1/100 dilution is written as 10-2 etc.) For example, if 1 ml of the 10-1 dilution from the A small portion of appropriate dilutions (depending last example were transferred to 9 ml of diluent, it on the estimated cell density of the original sample) is then would become a 10-2 dilution as follows. 1 spread onto agar plates to produce at least one countable _ V 1D 1 _ 1.0 ml X 10-1 _ _ _ _ _ D2 - - - - - 10 1 X 10 1 - 10 2 plate. A countable plate is one that contains between 30 and 300 colonies (Fig. 6.2). A count lower than 30 colonies V2 10 ml is considered statistically unreliable and greater than 300 is Spreading a known volume of this dilution onto an typically too many to be viewed as individual colonies. agar plate and counting the colonies that develop would In examining the procedural diagram (Fig. 6.1), you give you all the information you need to calculate the can see that the first transfer in the series is a simple original cell density (OCD). Below is the basic formula dilution, but that all successive transfers are compound for this calculation. CFU OCD = DxV CFU (colony forming units) is actually the number of colonies that develop on the plate. CFU is the preferred term because colonies could develop from single cells or from groups of cells, depending on the typical cellular arrangement of the organism. D is the dilution as written on the dilution tube from which the inoculum comes. V is the volume transferred to the plate. (Note: The volume is included in the formula because densities are expressed in CFU/ml, therefore a 0.1 ml inoculation [which would contain 1110th as many cells as 1 ml] must be accounted for.) 1 Permutations of this formula work with all necessary dilution calculations. For calculations involving unconventional volumes or dilutions, the formula is essential, but for simple tenfold or hundredfold dilutions like the ones 6.2 Countable Plate + A countable plate has between 30 and described in this exercise, the final compounded dilution in a series can be calculated simply by multiplying each of the simple dilutions by each other. 300 colonies. Therefore, this plate with approximately 130 colonies For example, a series of three 10-1 dilutions would yield a final dilution of is countable and can be used to calculate cell density in the original l0- 3 (10-1 X 10- 1 X 10- 1 = 10-3 ). Three 10-2 dilutions would yield a final sample. Plates with fewer than 30 colonies are TFTC ("too few to dilution of 10-6 (10- 2 X 10-2 X 10-2 = 10- 6 ). We encourage you to use count"). Plates with more than 300 colonies are TNTC ("too numer- whatever method is best for you. In time you will be doing the calculations ous to count"). in your head.  As you can see in the formula, the volume of original D Eight nutrient agar plates sample being transferred to a plate is the product of the D Screw-cap jar (and its lid) containing ethanol and a volume transferred and the dilution of the tube from which bent glass rod it came. Therefore 0.1 mL transferred from a 10-2 dilution D Hand tally counter contains only 10-3 mL of the original sample (0.1 mL X o Colony counter 10-2 ). The convention among microbiologists is to con- D 24-hour broth culture of Escherichia coli (this culture dense D and Vin the formula into "Original sample volume" 2 (expressed in mL). The formula thus becomes, ° will have between 2 X 10 7 and 2 X 10 1 CFU/mL) CFU OCD = Original sample volume Procedure The sample volume is written on the plate at the Refer to the procedural diagram in Figure 6.1 as needed. time of inoculation. Following a period of incubation, Appendix F provides an alternative procedure using digital the plates are examined, colonies are counted on the micropipettes and µL volumes. countable plates, and calculation is a simple division problem. Suppose, for example, you counted 37 colonies Lab One on a plate inoculated a plate with 0.1 mL of a 10-5 dilu- 1 Obtain eight plates, organize them into four pairs, tion. Knowing that this plate now contains 10-6 mL of and label them Ai, A2 , Bi, B2 , etc. original sample, calculation would be as follows. 2 Obtain five dilution tubes, and label them 1-5, CFU 37 CFU respectively. Make sure they remain covered until OCD = = -- - = 3.7 X 107 CFU/mL needed. Sample volume 6 10- mL 3 Aseptically add 9.9 mL sterile water to dilution tubes P.!111111 1 and 2. Cover when finished. Aseptically add 9.0 1:.111111 Application mL sterile water to dilution tubes 3, 4, and 5. Cover The viable count is one method of determining the density when finished. of a microbial population. It provides an estimate of actual living cells in the sample. 4 Mix the broth culture, and aseptically transfer 0.1 mL to dilution tube 1. Mix well. This is a 10-2 dilution. In This Exercise 5 Aseptically transfer 0.1 mL from dilution tube 1 to You will perform a dilution series and determine the dilution tube 2; mix well. This is a 10-4 dilution. population density of a broth culture of Escherichia coli. You will inoculate the plates using the spread plate 6 Aseptically transfer 1.0 mL from dilution tube 2 to dilution tube 3; mix well. This is a 10-5 dilution. technique, as illustrated in Exercise 1-5. As described in Figure 1.42, the inocula from the dilution tubes will be 7 Aseptically transfer 1.0 mL from dilution tube 3 to evenly dispersed over the agar surface with a bent glass rod. dilution tube 4; mix well. This is a 10-6 dilution. You will be sterilizing the glass rod between inoculations 8 Aseptically transfer 1.0 mL from dilution tube 4 to by immersing it in alcohol and igniting it. Be careful to dilution tube 5; mix well. This is a 10-7 dilution. organize your work area properly and at all times keep the flame away from the alcohol jar. Should a fire start 9 Aseptically transfer 0.1 mL from dilution tube 2 to plate A1 Using the spread plate technique, disperse in the jar, extinguish it by replacing the lid on the jar. the sample evenly over the entire surface of the agar. Repeat the procedure with plate A2 , and label./ Materials both plates "10-5 mL original sample," or simply Per Student Group "l0-5." D Sterile 0.1 mL, 1.0 mL, and 10.0 mL pipettes 10 Following the same procedure, transfer 0.1 mL D Mechanical pipettor volumes from dilution tubes 3, 4, and 5 to plates B, D Five sterile dilution tubes with caps C, and D, respectively. Label the plates accordingly. Allow the inocula to soak into the agar for a few D Flask of sterile normal saline minutes before continuing. 2 Some microbiologists refer to this as the " plate dilution." We prefer to use 11 Invert the plates and incubate at 35°C for 24 to 48 OSV in this introduction because it emphasizes what is really happening; hours. that is, OSV is the volume of the original sample deposited on the plate.  Lab Two 1 After incubation, examine the plates and determine the countable pair-plates with 30 to 300 colonies. Only one pair of plates should be countable. The remainder should be "too few to count" (TFTC) or "too numerous to count" (lNTC). 2 Count the colonies on both plates and calculate the average (Fig. 6.3 ). Various methods are available for counting. A simple way is to use a hand tally counter and a felt-tip marker. Mark each colony on the plastic base and simultaneously click the counter. 3 Record these in the table provided on the data sheet, page 473. (Note: You may have more than one pair of plates that is countable. For the practice, count all plates that have between 30-300 colonies, and try to identify which pair you have the most confidence in. If no plates are in the 30-300 colony range, count the pair that is closest.) 6.3 Counting Bacterial Colonies + Place the plate upside down on the colony counter. Turn on the light and adjust the magnifying 3 Using the formula provided on the data sheet, glass until all the colonies are visible. Using the grid in the background calculate the density of the original sample and as a guide, count colonies one section at a time. Mark each colony - -- record it in the space provided. with a felt-tip marker as you record with a hand tally counter. References 3 Other, more sophisticated counting methods are available. One uses an Collins, C. H., Patricia M. Lyne, and J.M. Grange. Page 149 in electronic pen that, when touched to the plastic Petri dish below a colony, Collins and Lyne's Microbiological Methods, 7th ed. Oxford, Boston: records a tally. There are also software systems that capture an image of Butterworth-Heinemann, 1995. the plate and then colonies are counted by the computer. Koch, Arthur L. Page 254 in Methods for General and Molecular Bacteriology. Philipp Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg, eds. Washington, DC: American Society for Microbiology, 1994. Postgate, J. R. Page 611 in Methods in Microbiology, Vol. 1. J. R. Norris and D. W. Ribbons, eds. New York: Academic Press, 1969. Date - - - - - - - - - - - - - - - - - - - - - - - - - - - Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - I was present and performed this exercise (initials) - - - - - - - - - - - - - Standard Plate Count (Viable Count) OBSERVATIONS AND INTERPRETATIONS 1 Enter the number of colonies on each countable plate. Only one pair of plates should be countable, but for practice, record all countable plates anyway. For all plates containing more than 300 colonies, enter TNTC ("too numerous to count"). For plates containing fewer than 30, enter TFTC ("too few to count"). If no plates are countable, use the TFTC plate closest to 30 colonies for practice with the calculations. Make a note of this in step 3 below. 2 Take the average number of colonies from the two (or more) countable plates and record it below. Plate A1 A2 B1 B2 C1 C2 D1 D2 Colonies counted Average# colonies 3 Calculate the original density in CFU/ml using the following formula: CFU OCD = Original sample volume I o,;g;oa1 deos;ty of E. coU ;, the bmth QUESTIONS When answering the following questions, assume all dilutions are in even powers of ten (unless told otherwise) and that 0.1 ml (100 µl) or 1.0 ml (1,000 µl) volumes were plated. 1 Suppose your professor handed you a test tube with 2.0 mL of an E.coli broth culture in it and told you to make a 10-1 dilution of the entire culture. Explain how you would do this. Show your calculations. 2 Suppose your professor handed you a test tube with 2.0 mL of an E.coli broth culture in it and told you to make a 10-2 dilution of the entire culture. Explain how you would do this. Show your calculations. SECTION 6 Quantitative Techniques 473  3 How would you produce a 10- 1 dilution of a 3 mL bacterial sample using the entire 3 mL volume? 4 How would you produce a 10 - 2 dilution of a 5 mL bacterial sample using the entire 5 mL volume? 5 You have 0.05 mL of an undiluted culture at a density of 3.6 X 106 CFU/mL. You then add 4.95 mL sterile diluent. What is the dilution and what is the final density of cells? 6 You have 0.3 mL of an undiluted culture at a density of 4.2 X 107 CFU/mL. You then add 2. 7 mL sterile diluent. What is the dilution and what is the final density of cells? IE 7 What is the dilution if 96 mL of diluent is added to 4 mL of a bacterial suspension? 8 What is the dilution if 75 mL of diluent is added to 25 mL of a bacterial suspension? 9 You were instructed to add 1.0 mL out of 5.0 mL of an undiluted sample to 99 mL of sterile diluent. Instead, you add all 5.0 mL to the 99 mL. What was the intended dilution and what was the actual dilution? 1Q Suppose you were instructed to add 0.2 mL of sample to 9.8 mL of diluent, but instead added 2.0 mL of sample. What was the intended dilution and what was the actual dilution? 414 MICROBIOLOGY: Laboratory Theory & Application Date-------------------------- DATA SHEET Lab Section - - - - - - - - - - - - - - - - - - - - - - - - 6-1 I was present and performed this exercise (initials) - - - - - - - - - - - - (continued) 11 Plating 1.0 mL of a sample diluted by a factor of 10- 3 produced 43 colonies. What was the original cell density in the sample? 12 Plating 0.1 mL of a sample diluted by a factor of 10 - 3 produced 43 colonies. What was the original cell density in the sample? 13 A plate with a sample volume of 10 - 7 mL produced 72 colonies. a. What was the original cell density? b. How many colonies should be on the plate inoculated with a sample volume of 1o-6 mL? c. How many colonies should be on the plate inoculated with a sample volume of 1 o-smL? 14 A plate with a sample volume of 1o-6 mL produced 259 colonies. a. What was the original cell density? b. How many colonies should be on the plate inoculated with a sample volume of 1o-smL? c. How many colonies should be on the plate inoculated with a sample volume of 10- 7 mL? 15 A nutrient agar plate that received 1,000 µL of a bacterial sample diluted by a factor of 1o-6 had 298 colonies on it after incubation. What was the original cell density? SECTION 6 Quantitative Techniques 475  16 You inoculated a nutrient agar plate with 100 µL of a sample diluted by a factor of 10 - 3 After incubation, you count 58 colonies. What was the original cell density? 17 A nutrient agar plate labeled 10 - 5 mL produced 154 colonies after incubation. a. What was the cell density in the original sample? b. What combination(s) of volumes and dilution factors could have been used to inoculate this plate? 18 A nutrient agar plate labeled 10 - 7 mL produced 62 colonies after incubation. a. What was the cell density in the original sample? b. What combination(s) of volumes and dilution factors could have been used to inoculate this plate? IE 19 The original cell density in a sample is 2. 79 X 106 CFU/mL. Which sample volume should yield a countable plate? (Express your answer as 1ox mL.) 20 The original cell density in a sample is 5.1 X 109 CFU/mL. Which sample volume should yield a countable plate? (Express your answer as 1ox mL.) 21 A sample has a density of 1.3 7 X 105 CFU/mL. a. What sample volume should yield a countable plate? b. Which two dilution tubes could be used to produce this sample volume? How? 22 A sample has a density of 7.9 X 10 9 CFU/mL. a. What sample volume should yield a countable plate? b. Which two dilution tubes could be used to produce this sample volume? How? 476 MICROBIOLOGY: Laboratory Theory & Application Date - - - - - - - - - - - - - - - - - - - - - - - - - - - DATA SHEET Lab Section - - - - - - - - - - - - - - - - - - - - - - - - - 6-1 I was present and performed this exercise (initials) - - - - - - - - - - - - - (continued) 23 You are told that a sample has between 2.5 X 103 and 2.5 X 105 cells!mL. Devise a complete but efficient (that is, no extra plates!) dilution scheme that will ensure getting a countable plate. 24 A sample has between 3.3 X 104 and 3.3 X 10 6 CFU/mL. Devise a complete but efficient (that is, no extra plates!) dilution scheme that will ensure getting a countable plate. 25 Two plates received 100 µL from the same dilution tube. The first plate had 293 colonies, whereas the second had 158 colonies. Suggest reasonable sources of error. 26 Two parallel dilution series were made from the same original sample. The plates with sample volumes of 1o-smL from each dilution series yielded 144 and 93 colonies. Suggest reasonable sources of error. SECTION 6 Quantitative Techniques 477