Lab 6 Bacteria Enumeration PDF

LAB 6 BACTERIA ENUMERATION Asst. Prof. Dr. Saharut Wongkaewkhiaw INTRODUCTION Enumeration of bacteria is defined as the process of determining the number of bacteria...

LAB 6 BACTERIA ENUMERATION Asst. Prof. Dr. Saharut Wongkaewkhiaw INTRODUCTION Enumeration of bacteria is defined as the process of determining the number of bacteria in a given sample. There are numerous reasons why researchers have to calculate the number of bacteria or compare the growing number of these microbes under certain specific conditions. It’s mainly essential and a part of routine work in food, water, cosmetics, pharmaceutical drugs, and microbiology labs in hospital. The knowledge of the numbers of these microorganisms helps the labs to determine whether the prepared food, water, or drugs is hygienic or not for consumption. Direct methods involve counting the microbes, while indirect methods involve estimation. Viable methods only count cells that are metabolically active, while total counts include dead and inactive cells. Categories of bacteria enumeration are divided into four categories: 1. Direct/Viable: It’s a standard plate count method in which repeated dilutions of a sample determine the number of viable bacteria cells in the given sample. 2. Indirect/Viable: This is a statistical inference about the microbial count based on growth patterns such as most probable number (MPN). It’s taken into consideration while enumerating the bacterial concentration in soil, industrial lab, or water. 3. Direct/Total: Dyes and fluorescent stains such as SYTO 9 and propidium iodide (PI) staining are used to make the bacterial cells visible using a fluorescent microscope to aid in counting the bacteria population in the given sample. It’s utilized while counting the numbers of bacteria in aquatic samples. 4. Indirect/Total: Spectroscopy is considered as indirect/total enumeration. With a spectrophotometer, the amount of light passed through the culture is used to estimate the number of microbes present. 20626104 General Microbiology for Dental Sciences 1 EXAMOLE TECHNIQUE FOR BACTERIAL ENUMERATION 1. Standard plate count (Direct/Viable) Bacteria can be present in thousands or millions in a sample, making it difficult to count their numbers. However, serially diluting the cultures makes it easier to determine the count. After serial dilution (Figure 6.1), the aliquots of the diluted sample are plated on an appropriate culture media. Then, the plates are incubated, after which the number of colonies formed is counted. This technique is also known as plate count or colony counts. Figure 6.1 An illustration of serial dilutions and plating techniques. The techniques that assist in standard plate count include “the spread plate and pour plate methods”. It’s essential to ensure that the plate is not crowded with bacterial colonies because, in such conditions, some cells might either not form colonies or fuse with each other resulting in erroneous results. Statistically, it is most valid to count colonies only on plates with 25 to 250 colonies. In this method, accurate determination of total cell numbers is only possible if each colony is formed of a single cell. However, it’s difficult for one to ensure such a case; that’s why the total numbers of cells reported using this method are termed Colony Forming Units (CFUs) rather than cell numbers. 20626104 General Microbiology for Dental Sciences 2 1.1 Spread plate The spread plate technique is a microbiological method used to isolate and enumerate bacteria or other microorganisms present in a sample. It involves spreading a diluted sample of microorganisms onto the surface of a solid agar medium in a petri dish. Figure 6.2 Schematic diagram of spread plate technique. 1.2 Pour plate The pour plate technique is a microbiological method used for the isolation and quantification of microorganisms in a sample. It involves mixing a diluted sample of microorganisms with a melted agar medium and then pouring the mixture into a petri dish. As the agar solidifies, microorganisms become evenly distributed throughout the medium, allowing for the growth of bacteria. Figure 6.3 Schematic diagram of pour plate technique. 20626104 General Microbiology for Dental Sciences 3  The pour plate technique and the spread plate technique are both microbiological methods used to isolate and quantify microorganisms from a sample, but they differ in how they distribute the microorganisms within the agar medium. Here are the key differences between the two techniques: 1. Distribution of microorganisms: - Spread plate technique: In the spread plate technique, a small volume of a diluted sample is spread evenly on the surface of a solid agar medium using a sterile spreader or glass rod. The goal is to separate individual microorganisms so that they form isolated colonies on the agar surface. - Pour plate technique: In the pour plate technique, a diluted sample is mixed with molten agar medium before pouring the mixture into a petri dish. The mixture solidifies as the agar cools, and this results in colonies growing both on the surface of the agar and within the agar itself. 2. Colony formation: - Spread plate technique: In this method, colonies grow only on the surface of the agar, as the microorganisms are deposited on the agar surface during the spreading process. Colonies are visible as distinct dots or clusters. - Pour plate technique: Colonies grow both on the surface of the agar and within the agar. Some colonies may be partially embedded in the agar, making them harder to distinguish, but this method allows for the isolation of microorganisms that might not have reached the surface in the spread plate technique. 3. Detection of low concentrations: - Spread plate technique: While the spread plate technique can detect low concentrations of microorganisms, it might be less effective at evenly distributing very low numbers across the agar surface. - Pour plate technique: The pour plate technique is particularly useful for detecting microorganisms present in low concentrations. The mixing step ensures even distribution within the agar, improving the chances of isolating colonies for enumeration. 4. Method of mixing: - Spread plate technique: The spreading is done manually using a sterile spreader or glass rod. The sample is placed on the agar surface and spread uniformly. 20626104 General Microbiology for Dental Sciences 4  - Pour plate technique: The mixing of the sample and agar occurs before pouring the mixture into the petri dish. The mixture is mixed by gently swirling or pipetting to ensure even distribution. Both techniques have their advantages and are suited to different situations. The choice between the two depends on factors such as the concentration of microorganisms in the sample, the type of agar being used, and the specific goals of the microbiological analysis. Advantages of plate count method; - Feasible for enumerating bacteria in a given sample. - Relatively simple to use and more sensitive than turbidimetric measurement. - It allows positive identification of the organism counted. - It’s easy to count small numbers of bacteria in a sample using this method. Limitations of plate count technique; - It’s a time-consuming process where every stage, including dilutions, platings, incubations, and media preparation. - Only living cells develop colonies; therefore, this method can only count them. - Clumps or chains of cells can develop into a single colony, leading to erroneous results. - A colony can only arise from organisms that can grow under the provided conditions. This can lead to miscalculation. 2. Turbidimetric measurement (Indirect/Total) Turbidity measures the loss of intensity of transmitted light due to the scattering effect of suspended particles (Figure 6.4). It’s simply the amount of cloudiness or turbidity in the sample. It can be caused by silt, sand, mud bacteria, and other microbes or chemical precipitates. The turbidimetric method is a quick and efficient method to measure and estimate the number of bacteria in a given sample. The method is most preferable when large numbers of cultures are required to be counted. When bacteria are mixed in a liquid medium, it creates a colloidal suspension that blocks and reflects light as it passes through the culture. The light absorbed by bacterial suspension will estimate the concentration of bacteria in the given sample. 20626104 General Microbiology for Dental Sciences 5  You must note that “the light absorbed by the culture is directly proportional to the cells’ concentration.” Figure 6.4 An illustration of turbidity measurement using a spectrophotometer. Even though the turbidity measurement technique is faster than the standard plate count, it is initially required to match the measurement values with cell numbers, which requires lab personnel to combine the standard plate count method with the turbidity technique. The procedure followed include: - First, the turbidity of different concentrations of a given bacterial species in a culture medium is determined. - Using standard plate count, the number of viable microorganisms per milliliter of the samples is estimated. - A standard curve is drawn, with turbidity or optical density readings corresponding to specific numbers of living organisms. 20626104 General Microbiology for Dental Sciences 6 Figure 6.5 Using light scatter to kinetically record changes in cell number. Once drawn, the standard graph can be directly used for subsequent turbidity measurement and calculating the number of viable bacterial cells (Figure 6.5). The method utilizes instruments such as a spectrophotometer or colorimeter to measure the turbidity in the given sample. Advantages of turbidimetric measurement; - It’s a faster procedure than the standard plate count technique. - It can be done without destroying the sample. Limitations of turbidimetric measurement; - For the estimation, it requires a high amount of bacterial cells—about 100 million cells per milliliter. - If the mass of bacteria increases more than normal in the sample, they mask the light passed through the cuvette, resulting in inaccurate calculations. - It does not distinguish between living and dead cells. 20626104 General Microbiology for Dental Sciences 7 3. Direct microscopic count (Direct/Total) The direct microscopic count is used for quantitative enumeration of bacteria liquid samples. It is done by spreading a measured volume of the sample over a predetermined area of a slide, counting representative microscopic fields, and transforming the average values into appropriate volume-area factors. The counting chambers used for the procedures such as Petroff-Hauser chambers (Figure 6.6). The Petroff-Hauser is a thick microscope slide with a chamber in the middle of 0.02 mm (1/50 mm) deep, and it also has improved Neubauer rulings and an etched grid in the chamber. The ruling is the centerline of a group of three squares in which cells are counted. Figure 6.6 An illustration of Petroff-Hauser counting chamber. A single drop of culture is applied in this counting method using a Pasteur pipette. The cell numbers in the given sample are counted directly in 10-20 high microscope fields. Based on the average number of cells per field, the number of bacteria per milliliter of the original sample can be obtained. Concentration of the cells can be calculated by using the average no. of bacteria the avg. number of bacteria in these squares. 20626104 General Microbiology for Dental Sciences 8  - There are 25 squares covering a part of area of 1 mm2, then the entire number of bacteria in 1 mm2 of the chamber is (number/square) (25 squares). The chamber is 0.02 mm deep and thus, bacteria/mm3 = (bacteria/square) (25 squares) - The amount of bacteria per cm3 is 103 times this value. For example, imagine the average count per square is 28 bacteria: bacteria/cm3 = (28 bacteria) (25 squares) (50) (103) = 3.5 X 107. Advantages of direct microscopic count - Direct counting procedures are rapid, simple, and easy. - You can observe the morphology of the bacteria while counting the numbers. Limitations of direct microscopic count - They do not discriminate between living and dead cells. - You can’t observe small cells, so they can be missed during counting. - This method cannot be used with cell suspensions of low density, i.e.,