PAG 7 2 Dilution plating to determine microbial

PAG 7. 2 Dilution plating to determine microbial density in liquid culture

Dilution plating to determine microbial density in liquid culture Aim To find the density of viable bacterial cells in two cultures of Bacillus subtilis by dilution plating. • Scientists use a number of different methods to determine the number of micro-organisms that are present in a given population. • The method we are looking at is serial dilution of the bacteria and plating the diluted bacteria on media that supports the growth of the micro-organisms. • You will make serial dilutions of 2 liquid cultures of Bacillus subtilis and then make spread plates: 24 h culture of Bacillus subtilis and 48 h culture of Bacillus subtilis • By diluting a culture of bacteria, spreading it on an agar plate and incubating that plate it is possible to count individual bacterial colonies. • Each colony arises from a single bacterial cell so you will then be able to calculate an estimate of the number of viable bacterial cells in the original culture. • The method is time consuming, but provides statistically accurate and repeatable results. • This method is also the ideal method for enumerating(establishing the number of) microorganisms in a given population because it only identifies the living organisms in that population. • You will make serial dilutions of a solution containing an unknown number of bacteria, plate these bacteria and determine the total number of bacteria in the original solution by counting the number of colony forming units and comparing them to the dilution factor.

Culturing microorganisms • To culture they need correct nutrients, temperature, p. H and oxygen • The nutrient medium and can be a broth or agar • Both must be kept very sterile • You used agar in AS and broth in A 2.

What would your prediction be? Aim To find the density of viable bacterial cells in two cultures of Bacillus subtilis by dilution You will make serial dilutions of 2 liquid cultures of Bacillus subtilis and then make spread plates: 24 h culture of Bacillus subtilis and 48 h culture of Bacillus subtilis • The density of bacteria in the older culture should be significantly greater as bacterial cells have divided more (time) • When conditions are favourable such as the right temperature and nutrients are available, some bacteria like Escherichia coli can divide every 20 minutes. • This means that in just 7 hours one bacterium can generate 2, 097, 152 bacteria. After one more hour the number of bacteria will have risen to a colossal 16, 777, 216 • https: //www. bbc. com/education/guides/z 243 g 82/revision/2 • Draw The standard growth curve of a microorganism in a closed culture • In a closed system what are the limiting factors on exponential growth in a bacterial culture?

In a closed system what are the limiting factors on exponential growth in a bacterial culture? Nutrients available • Oxygen levels • Temperature • Build up of waste • Change in p. H (carbon dioxide) • (See page 596) • Why are there such clear differences between theoretical growth curve of a bacterial colony and the actual growth curve in a closed culture? • 5 marks • In large closed culture nothing gets in or out (1); • Initially, growth can be at theoretical rate as no factors are limiting (1); • As culture continues, numbers increase, food and oxygen are used up and waste products build up often affecting p. H (1); • Microorganisms run out of food or oxygen, are inactivated by p. H changes affecting enzymes or poisoned by waste products (1); • So whilst theoretical growth curve is exponential, real growth curve reaches a peak, plateaus, and declines (1). •

Direct cell counts may be divided into 1. Total counts = which include both living cells and dead cells 2. Viable counts = which count living cells only In practice, it is never possible to count whole populations of microorganisms. Instead, the cells in a very small sample of culture are counted, and the result multiplied up to give a population density in organisms per cm 3 of culture. Even then, the population density is likely to be so high that cell counts are usually made in known dilutions of the culture, usually in 10 -fold steps. This is known as serial dilution. What is a serial dilution? It is a repeated stepwise dilution of a stock solution of a known concentration. It is usually done by factors of 10, to produce a range of concentrations. Can be done when concentration of initial solution is unknown as give relative concentrations (you used at AS looking at different concentrations of catalase? ? )

Serial dilutions • You will now make a serial dilution so that you have samples from each culture diluted 10, 1000 and 10, 000 times. • Decide on the volume of sterile LB broth you will put into each of your sterile Bijou bottles and the volume of culture that you will need to add to each. • See diagram: • Volume of LB broth 9 m. L • Volume of Bacillus subtilis culture 1 cm 3 = 1 m. L You are going to set up: • Volume of LB broth: 9. 9 m. L • Volume of Bacillus subtilis culture : 0. 1 m. L

Calculating serial dilutions • You multiply the original concentration by the dilution factors for each dilution. • Explanation: • A serial dilution is any dilution in which the concentration decreases by the same factor in each successive step. • In serial dilutions, you multiply the dilution factors for each step. • The dilution factor or the dilution is the initial volume (Vi) divided by the final volume (Vf) • DF=Vi/Vf • For example, if you add a 1 m. L sample to 9 m. L of diluent to get 10 ml of solution, • DF=Vi/Vf = 1 ml/10 ml=1/10. This is a 1: 10 dilution.

Example 1. What is the dilution factor if you add 0. 2 m. L of a stock solution to 3. 8 m. L of diluent? DF=Vi/Vf • Vf = 0. 2 m. L + 3. 8 m. L = 4. 0 m. L • DF= Vi = 0. 2 m. L 1 Vf 4. 0 m. L 20. • This is a 1: 20 dilution 2. If you did the above dilution 4 x what would be the final dilution factor? You would transfer 0. 2 m. L from tube 1 to 3. 8 ml of diluent in tube 2 and mix, and repeat until you have 4 tubes DF= 1 x 1 x 1 16 20 20 0000 • If the concentration of the original stock solution was 100 µg/µL, the concentration in Tube 4 would be • 100 µg/µL × 1 = 6. 25 × 10⁻⁴ µg/µL 160000

Chemicals and Safety • ASEPTIC TECHNIQUES? ? ? • Health and Safety • Ethanol is highly flammable and you have a naked flame on your bench. Great care is therefore needed to minimise the risk of fire. Have only a small volume of ethanol in your wide dish, keep it covered when not in use and well away from the Bunsen burner. • Wear a lab coat and goggles when working with microorganisms. • Cover any skin cuts or abrasions. • All spills must be immediately disinfected using 1% Virkon® solution left in place for 10 minutes

Method: Part 1 – diluting the cultures and spreading the plates Note: At all times in this investigation, a roaring Bunsen flame should be burning on the bench. This is needed for sterile working. However, it is a fire hazard. Keep your dish of ethanol well away from the Bunsen burner and keep the dish covered when not in use. • Wipe your bench with antibacterial spray and a paper towel. Pour 1% Virkon® solution into your tray to cover the bottom with a thin layer, leave in place for 10 minutes and then wipe away. • Take five sterile agar plates and label them: 24 h 100, 24 h 10 -1, 24 h 10 -2, 24 h 10 -3 and 24 h 10 -4 • • Take the other five sterile agar plates and label them: 48 h 100, 48 h 10 -1, 48 h 10 -2, 48 h 10 -3 and 48 h 10 -4 • Take four sterile Bijou bottles and label them: 24 h 10 -1, 24 h 10 -2, 24 h 10 -3 and 24 h 10 -4 • Take the other four sterile Bijou bottles and label them: 48 h 10 -1, 48 h 10 -2, 48 h 10 -3 and 48 h 10 -4 • You will now make a serial dilution so that you have samples from each culture diluted 10, 1000 and 10, 000 times. Decide on the volume of sterile LB broth you will put into each of your sterile Bijou bottles and the volume of culture that you will need to add to each.

Part 2 – observing the colonies and calculating original densities After one day, look at the plates but do not open them. To record your observations, draw and annotate each of your ten spread plates. In each set of five plates, identify one where you can see individual colonies. Count the number of colonies on the plate you have chosen from each set and record this number and the dilution factor for that plate. Use your data to calculate an estimate of the density of viable bacteria in each of the two original cultures and record your results using appropriate units.

Counting colonies:

v. After incubation, the number of colonies on a dilution plate showing between 30 and 300 colonies is determined. v. A plate having 30 -300 colonies is chosen because this range is considered statistically significant v. If there are less than 30 colonies on the plate, small errors in dilution technique or the presence of a few contaminants will have a drastic effect on the final count. (too few to count (TFTC). v. Likewise, if there are more than 300 colonies on the plate, there will be poor isolation and colonies will have grown together. (too numerous to count (TNTC).

CFU stands for Colony Forming Units, a term in microbiology used to quantify how many bacteria are present in a solution • Perform a preliminary count of each dish once the bacteria have had time to incubate, usually one or two days. • You want to count only individual colonies, which should be distinct, isolated dots, not a whole blob of different colonies grown together. • Choose the plate which has more than 30 of these colonies but less than 300. • v. Count the number of individual colonies. This is the CFU number of your dilution -- you will have to perform a simple calculation to determine the CFU of the original sample. v. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to agar plate. v. To compute the number of CFU/m. L, use the formula: c = concentration, CFU/m. L § n = number of colonies § d = dilution blank factor § S = volume transferred to plate. §

CFU Calculation Example § You count 46 colonies on your plate § You put 1 ml of bacterial culture into 99 ml of saline and plated 0. 1 ml § Dilution 1/100 § CFU= 46 (n) 1/100 (d) x 0. 1 (S) v. Calculate the number of bacteria (CFU) per milliliter or gram of sample by dividing the number of colonies by the dilution factor multiplied by the amount of specimen added to agar plate. v. To compute the number of CFU/m. L, use the formula: § c = concentration, CFU/m. L § 46 (n) o. 01 (d) x 0. 1 (S) = 0. 001 = 46 (n) 0. 001 = c = 46000 CFU/ml WATCH https: //www. youtube. com/watch? v=e. Jr 3 yt 0 EREg § n = number of colonies § d = dilution blank factor § S = volume transferred to plate

Answers to extension questions • What other methods could be used to find the density of bacteria in a culture? Why might some of these methods, even when carried out carefully and accurately, give results different from the dilution plating method? Microscopy could be used. A haemocytometer, or similar suggestion of a standardised method, would be suitable. This method would identify cells but could include dead cells, leading to an overestimate compared with the dilution plating method. Staining can be used to identify living versus dead cells and this will bring the results of the two methods closer together although there will still be some fraction of cells that are alive, and hence able to exclude the stain, but not able to divide. Turbidity of the culture can be used, once a calibration curve has been produced from a known set of cultures. The % transmission of light through the culture can be measured with a colorimeter and the density of the unknown culture inferred from the calibration curve. This method takes no account of viability of cells so will give an overestimate, especially in old, high density cultures. • When observing your spread plates, how can you be confident that all the colonies you see are Bacillus subtilis? The quickest check to be made is simply colour and morphology of colonies. All colonies should look the same. Any different colonies are almost certainly the result of contamination. Microscopic observation of samples from any suspect colonies, using identification aids such as Gramstaining, could confirm this. • One of the cultures you were provided with was 24 h older than the other. Use your results to calculate the percentage increase in cell number that has taken place in the 24 h that separates the two cultures. Answers to this will depend on the results obtained by the students. This could be discussed by the whole class to see how the range of answers varied across the group. • Assuming that the bacteria have been dividing at a constant rate throughout those 24 h, how long does it take each cell to divide? Answers to this will depend on the results obtained by the students, but what would you do?

Compare the processes of culturing bacteria in Broth and on agar: 6 Marks • Both provide nutrients, suitable p. H, moisture etc (1); • both need to be maintained at optimum temperature for growth (1); • both must be kept sterile until inoculated with microorganisms (1); • both can be shaken at intervals to aerate it (1); • agar plates remain closed once made up (1); • broth is mixed with known volumes of culture medium (1); • agar plates inoculated using sterile wire loop and culture medium (1); • numbers in broth counted using turbidity, serial dilutions, and microscope graticules (1); • numbers on agar calculated using colony counting (1) • (6 max, must be at least two similarities and differences)

Investigating factors which affect the growth of microorganisms: serial dilutions and bacterial counting. • What other factors could you investigate • Remember you have to apply your understanding. • Set up identical colonies in different temperatures • Set up serial dilutions of nutrients • Set up serial dilutions of p. H

Make a flow chart to show you would calculate the affect of a factor on bacterial growth using serial dilutions and agar 6 marks Could be done in a number of ways (max 6) e. g. , 1. set up series of cultures identical in every way EXCEPT for factor you are investigating e. g. , temperature, presence of a nutrient or antibiotic etc 2. Culture for several days 3. For each culture carry out a serial dilution 4. Plate each dilution, making streaks on agar plate with sterile inoculating loop and label carefully to show which original culture it has come from and what dilution factor is 5. Repeat for all original cultures 6. Culture agar plates for 5 days at 20°C 7. For each factor find plate with number of bacterial colonies that can be counted. 8. Work out original number of bacteria using dilution factor 9. Compare results across different cultures to see effect on bacterial growth of factor being investigated. •