Microbiology sheet (6)

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1 Microbiology sheet (6) Made by marah marahleh corrected by : abd. Salman

2 DATE :9/10/2016 Microbial growth / control of microbial growth 1 The method of counting bacteria is divided into: 1) direct 2) indirect Viable cell counts: Plate counts: Serial dilutions put on plates CFUs form colonies Plate count is one of direct methods for counting bacteria

3 2 There is two types of plate count: 1) the pour plate method plate method 2) the spread The difference between them is that I have to dilute the sample whether the sample is water, juice, milk etc.... We don t usually use this method for urine sample. Dilution in bacteria is log dilution (10^-1, 10^-2, ) In the growth curve in the previous lecture e used time vs. log number of bacteria not the time vs. the number of bacteria

4 So in the first type (the pour plate method) we used *antipetri dish containing 1 ml of *every solution with 13 ml of melted agar and mix it then leave it to dry for *30 minute and put it upside down in the incubator 3 (2:39) the number of colony used in the plate more than 30 and less than 300 (30-300) Some food microbiologists use from 25 to 250 In the second type we have petri dish and take 0.1 ml for each solution* then we spread the liquid with a spreading glass over the surface of the agar then leave it to dry and put it in the incubator then we count the number of bacteria When we count the colony forming unit = number of colony * dilution factor but not to the power of minus because we want to return to the original number of bacteria in the sample (4:50) the device is a colony counter which makes the plate larger so it is easier for the technician to count the number of colonies and it is supposed to give us similar number in any plate we use Why? Because if the number is small we multiply it by high dilution factor and vice versa

5 So in the first type we use one ml and in the second we use 0.1 ml and spread, it to even it out We should multiply it with correction factor 10 (number of colonies *10) Some of the disadvantages of this method is that some bacteria is sensitive to heat and the melted agar temperature is 55C also some bacteria will grow on the subsurface (beneath the actual surface) So the obligatory aerobic bacteria cannot survive these conditions 4 Direct microscopic count: Counting chambers (slides) for microscope A specially designed slide called a petroff-hausser cell counter is used in direct microscopic counts. -motile bacteria are difficult to count by this method -Dead cells are bout as likely to be counted as live ones. This method is also direct and use counting chambers.this method calculate the total number of bacteria (dead and viable cells) Viable cells can divide and form colonies unlike dead cells (cannot divide so they do not form colonies) Filtration is the method of choice for low counts M.O. Membrane filters for fluids.

6 Pore size for bacteria: m Pore size for viruses: 0.01 m 5 At least 100 ml of water are passed through a thin membrane filter whose pores are too small to allow bacteria to pass. Filtration is a direct method used with low concentration of M.O (microorganism) In this method we use membrane filters which have pores of specific size so the fluid go through the pores but the bacteria cannot and stays on the surface of the membrane, it cannot be used with high concentration because the bacteria is in high concentration it will block the pores For the bacteria the pore size is ( micro m in diameter) We take the filter and put it on petri dish (selective media) and the water will transport from the media to the membrane filter and after 24 hours we will obtain colonies of bacteria that we can count If the sample is water and contain heavy metals cannot used this method because the heavy metals will block the pores Only viable cells is counted in this method

7 Indirect methods Spectrophotometry to measure turbidity OD is function of cell number 6 Spectrophotometry is an indirect method Spectrophotometry device is used to measure absorbance In the second test tube most of the light will hit the bacteria and will be scattered and some of the light will reach the device Direct relationship between absorbance and the number of bacteria Indirect relationship between transmission and number of bacteria

8 7 The most probable number (MPN) method - Another method for determining the number of bacteria in a sample is the most probable number (MPN) method - This statistical estimating technique is based on the fact that the greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution series. - It is useful when the growth of bacteria in a liquid differential medium is used to identify the microbes (such as coliform bacteria, which selectively ferment lactose to acid, in water testing). تعتمد على عامل التخفيف بنعمل تخفيف لمرحلة اعداد البكتيريا قليل جدا Used mostly for water samples to identify microbes usually coliform bacteria Coliform bacteria is: 1) E.coli 2)klebsiella 3) enterobacteria And their presence is an indicator for fecal contamination in the water, the more the contamination of the sample the more dilution is needed We use selective media that depends on trait of coliform bacteria, coliform bacteria produce acid and gas during fermentation, the acid will change the color of the media and the gas will be collected in small tubes called Durham tubes 8

9 We take three sets of test tube each set composed of five test tube that contain 10 ml of selective media The empty spaces in the tubes are gas First test 10 ml of water sample Second test 1 ml of water sample Third test 0.1 ml of water sample The whole water samples should be 100 ml 4 positive result ( contain gas ) 3 positive result 1 positive result After incubation will see change in color and gas formation We take the positive result (in our case ) and compare it with the standard table (slide 33) 9

10 The WHO divide water purity to 4 groups : <2 pure water, 2-8 acceptable 8-40 doubtful, >40 swage water Q: will the bacteria grow in the time needed to make the tests? No because microbiology departments always have these test ready all they have to do is obtain the samples Metabolic activity - indirect way to estimate bacterial numbers is to measure A population's metabolic activity (acid or CO2, is in direct proportion to the number of bacteria present). 10

11 Indirect method The bacteria produce during metabolism end product like gas and these products is compared to standard values (low number of bacteria = 10 colonies or none at all) The product of metabolism is direct proportion to number of bacteria Ex: CO2 for a sample of bacteria is measured and compared to CO2 concentration of standard sample that contain low number of bacteria or none Dry weight -For filamentous bacteria and molds, the fungus is removed from the growth medium, filtered, and dried in a desiccator. It is then weighed Used usually for filamentous bacteria (bacteria between fungi and bacteria it looks like fungi on plates but it is actually bacteria The dry weight is indirect proportion with number of bacteria Measuring Microbial Growth Overview Direct Method Methods Plate counts MPN activity Direct microscopic count Indirect turbidity metabolic dry weight Filtration 11

12 Control of microbial growth We talked previously about the chemical and physical factors to grow bacteria here we will use these factors to kill, control and prevent contamination Alcohols and antibiotics are some of the chemical means to kill bacteria Temperature is one of the physical factors, any increase in the temperature might affect the bacteria to the point of killing it.(because in our body we deal with mesophile bacteria) Terminology Sepsis: Characterized by the presence of pathogenic microbes in living tissues or associated fluids. (Contaminated) Asepsis: absence of significant contamination. (Not contaminated) Aseptic surgery techniques prevent microbial contamination of wounds. Antimicrobial: chemicals, expected to destroy pathogens but not to achieve sterilization. Disinfectant: used on objects (reduce the number of viable microorganisms) kill viable cells only does not achieve sterilization. We use alcohol as disinfectant and antiseptic as well. تكون للطاولة مثال)تطهير( اما antiseptic فتكون مثال للجلد Disinfectant Antiseptic: used on living tissue, destroys or inhibits the growth of microorganisms does not achieve sterilization. Nosocomial Infection (Hospital Acquired Infection) an infection that is contracted from the environment or staff of a healthcare facility. (Mostly acquired from the staff normal flora 12

13 Every person have normal flora and bacteria which he have biological tolerance to so it does not cause any disease to him but it might cause infection and be pathogenic to another person who does not have the same normal flora and thus does not have biological tolerance to it اخماج: sepsis Anti: against Sterilization: A defined process used to render a surface or product free from viable organisms, including bacterial spores. Sterilization: absence of any microorganisms (no viruses, no bacteria nothing ) Biocide: A chemical or physical agent, usually broad spectrum that inactivates (kill) microorganisms. Chemical biocides include hydrogen peroxide, alcohols, bleach, cycloheximide, and phenols Physical biocides include heat and radiation. Fungicide, Virucide, Germicide, bactericide -Cide = kill - Broad spectrum = kills gram (-) and gram (+) and spore forming bacteria -fungicide: kills fungi - virucide: kills viruses - germicide: kills germs (general concept) - Bactericide: kills bacteria Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms) lowering of microbial counts to be accepted by public health regulation. 13

14 Antibiotics: Naturally occurring and synthetically derived organic compounds that inhibit or destroy selective bacteria, generally at low concentrations First antibiotics were natural but know mostly are synthetic. Ex: penicillin. We use antibiotic at low concentration because at high concentration it becomes toxic. There is two kind of antibiotics: Bacteriostatic: Inhibits bacterial reproduction (affect one stage of bacterial growth ex: nucleic acid and protein synthesis). مثبطة وليست قاتلة Bactericidal: Kills bacteria (ex: penicillin). 14

15 Clostridium botulinum is kind of bacteria that cause severe food poisoning (lethal) Can lead to paralysis (disease called botulism). In the production of canned food microbiologists take random samples from canned food and put it in an incubator and check it after 2 weeks so we decide if we put it in supermarkets or no. Any bump in canned food would be caused by gases produced by bacteria. 15