BIO 351 Lab 3 & 4 Techniques for Isolation of Pure Cultures

BIO 351 Pg.16 LAB 3 and 4 Techniques for Isolation of Pure Cultures – The Bacterial Growth The Streak Plate General: In nature microbial populations do not segregate themselves by species but rather exist in a mixture of many ot...

BIO 351 Pg.16 LAB 3 and 4 Techniques for Isolation of Pure Cultures – The Bacterial Growth The Streak Plate General: In nature microbial populations do not segregate themselves by species but rather exist in a mixture of many other cell types. In the Laboratory, these populations can be separated into pure cultures. These cultures contain only one (1) type of organism. Why do we want to isolate the organism and how do we do it? We want to find an isolated colony, select it by touching it with a sterile loop and transferring it to a separate media plate to grow as a pure culture. We can then grow and add antibiotic sensitivity disks to see which ones can kill the bacteria, for the purpose of treating patients. The disks are paper type dots containing small sample of the antibiotic drug. If the organism is killed there is a “zone of inhibition” surrounding the bacterial CFU. So the result Is either S=Sensitive (inhibits the bacterial growth) or R=Resistant to the antibiotic, shows no effect or zone surrounding the bacteria. The Streak Plate is a method enabling rapid qualitative isolation of discrete colonies. In order to obtain the discrete colony, it requires reducing or “spreading out” the number of organisms from the inoculum. Initially placed in quadrant “A” and streaking it out according to the procedure below. The technique consists of creating or spreading the inoculum into 4 parts or quadrants, with the 4th quadrant or area “D” having the widest streaks and hopefully containing isolated CFU’s: PROCEDURE: LABEL the petri dish on the agar side as to your initials (or patient’s), organism if know and date of inoculation: REMEMBER TO USE ASEPTIC TECHNIQUE! 1. Flame a loop to make it sterile, and cool it by touching an unused part of the agar (near the periphery of the plate). With the sterile loop, place a loopful of culture on the agar surface in Area-A, then drag it rapidly several times (5-6) across the surface of Area-A 2. Reflame and cool the loop, and turn the Petri dish 90 degrees. Then touch the loop to a corner of the culture in Area-A and drag it several times (5-6) across the agar in Area-B. The loop must never enter Area-A again 3. Reflame and cool the loop and again turn the dish 90 degrees. Streak Area-C in the same manner as Area-B 4. Without reflaming the loop, again turn the dish 90 degrees and then drag the culture from a corner of Area-C, across Area-D, using a wider streak. Don’t let the loop  BIO 351 Pg.17 touch any of the previously streaked areas. NOTE: The flaming of the loop helps to further dilute the bacteria. Lab 3 Exercise: Prepare a Streak Plate of liquid “mixed-culture” of 3 bacteria on TSA agar. Label your plates with your initials, date and MC for mixed culture. 1. Serratia marcescens – 1 part produces a red color 2. E. coli – 1 part – produces a white/grey color 3. Staph aureus (or micrococcus luteus)-10 parts-produces yellow color Goal: To see if you can isolate discrete CFU (colonies) of each of the 3 bacteria in Area 4 or Area “D” of the plate after incubation to obtain a pure culture of each bacteria based on color. _____________________________________________________________________ LAB 4: Look at the streak plates to see if you were able to isolate individual CFU in quadrant D or area 4 of the incubated petri dish. Students should see the 3 different colors of CFU’s: red, yellow and white/grey for the 3 bacteria that was contained in the mixed culture: Serratia marcescens: red color E, Coli: white/grey color Micrococcus luteus: yellow color The goal is achieved if you are able to isolate one CFU for each organism for the purpose of further “sub culturing” that organism to its own plate. _______________________________________________________________________ Lab 3: Microbial Growth – When we are discussing microbial growth we are referring to the number of cells, not the size of cells. Microbes that are “growing” are increasing in number, accumulating into colonies (groups of cells large enough to be seen without a microscope) of hundreds of thousands of cells populations of billions of cells. Although individual cells approximately double in size during their lifetime, this doesn’t compare with size increases seen in the lifetime of other plants and animals. Microbes will grow depending upon their environment and as such will grow slower in a nutrient-poor grow slowly and form biofilms (a microbial community that usually forms as a slimy layer on a surface). Biofilms are frequently sources of health care-associated infections. Microbial populations can become incredibly large in a very short time. By understanding the conditions for growth, we can learn how to control the growth of microbes that cause diseases and food spoilage. We also learn how to grow microbes that we wish to study. First, we need to review and understand the normal bacterial growth curve which is illustrated in Figure 1 on page 123 of your Lab Manual and here on the next page: BIO 351 Pg.18 Principle: Bacterial populations growth studies requires the inoculation of viable cells. The viable cells provided are E. coli in a sterile broth medium. Students will prepare dilutions of the pure culture and then transfer different volumes of the dilutions to sterile empty Petri dishes which will later have liquid (50 C.) liquid agar added to these plates. They will then be gently mixed by swirling the plate and left to harden. The plates will then be incubated by the technician in preparation for direct bacterial CFU counting the following week. Population Growth Curve: The four stages of a typical growth curve: 1. Lag Phase: during this stage the cells are adjusting to their new environment. Cellular metabolism is accelerated, resulting in rapid biosynthesis, primarily enzymes, in preparation for the next phase. Cells are increasing in size there is no cell division and therefore no increase in numbers 2. Logarithmic (log) Phase: under optimal nutritional and physical conditions, the physiological robust cells reproduce at a uniform and rapid rate by transverse binary fission. Thus, there is rapid exponential increase in population which doubles regularly until a maximum number of cells is reached. The time required for the population to double is the generation time. The length of the log phase varies, depending on the organisms and the composition of the medium. The average time of this phase is 6-12 hours 3. Stationary Phase: During this stage, the number of cells undergoing division is equal to the number of cells that are dying. Therefore, there is no increase in cell number, and the population is maintained t its maximum level for a period of time. The primary factors responsible for this phase are the deletion of some essential metabolites and the accumulation of toxic acidic or alkaline end products in the medium  BIO 351 Pg.19 4. Decline, or death phase: Because of the continuing depletion of nutrients and buildup of metabolic wastes, the microorganisms die at a rapid and uniform rate. The decrease in population closely parallels its increase during the log phase. Theoretically, the entire population should die during a time interval equal to that of the log phase. This does not occur, however, since a small number of highly resistant organisms persist for an indeterminate length of time Direct Method of Counting Bacterial Growth Lab 3: Refer to Lab Manual Pg.126 for illustration of this Lab Exercise. The experiment was set up in Lab 5 and involved transferring 1 mL of a pure liquid culture of E. coli to a 99 mL bottle of sterile water, Labeled bottle ‘A”. This bottle was then vigorously shaken to mix the fluid and then transfer 1 mL to another 99 mL bottle of sterile water, labeled bottle “B”. This bottle too was thoroughly mixed and then we transferred 1 mL of bottle “B” mix to a third bottle of 99 mL sterile water, labeled, bottle “C” – it too was thoroughly mixed. We then labeled 4 empty, sterile Petri dishes: A, B C and D. Starting with bottle “B” we pipetted 0.1 mL to plate “A” and 1.0 mL of the mixture to plate “B”. With a clean pipette, we then transferred 0.1 mL of bottle “C” mixture to plate “C” and 1.0 mL of it to plate “D”. We then added liquid nutrient agar incubating at 50 C. to each plate separately and swirled the mixture. The plates were covered, all to solidify and then incubated by the technician. Lab 4: To count the bacterial colonies or “Colony forming units” (CFU) we utilize a Quebec Counter which provides a magnified light source that the entire petri dish sits on for examination. The rule is we can only count a plate that contains between 30 and 300 CFU. Calculation of the Dilution Factors (DF) for each bottle and plate is essential to our final calculation. We determined the following DF calculations: Bottle A is 1/100 ----transferred to Bottle B x100 it becomes 1/10,000, bottle C is then 100x this and1 bottle C becomes 1/1,000,000 Starting with Bottle “B”: We transfer 0.1 mL to Plate A is DF is 1/100,000, then 1.0 mL to plate B, DF is 1/10,000 (the same as bottle “B”). Then we transfer 0.1 mL of Bottle “C” to plate C, the DF is 1/10 million, then 1.0 mL from Bottle “C” to plate D, the DF of plate D is 1/1,000,000 (the same as bottle “C”) To calculate the total bacteria, we take the # of CFU counted on plate x DF = Total Number Rules: 1. When counting a plate that has a total CFU count of between 30-300, we only count CFUs that are in free spaces on the Quebec counting screen, none that fall on the lines, only those seen above and below the lines 2. When making a dilution of a dilution, you must factor for the new dilution. 3. Use the calculated DF for plate being counted times the # of CFU to determine the total count. BIO 351 Lab #5 Pg. 20 Cultivation of Microorganisms: Nutritional and Physical Requirements for Growth As all other living organisms, microorganisms require certain basic nutrients and physical factors for the sustenance of life. However, particular requirements vary greatly. Understanding them is necessary for successful cultivation or growth in the laboratory. Another key influence for the control of microbial growth is the physical agents of control: Chemical Agents and Osmotic Pressure. I Nutritional and Chemical Requirements for Growth: Carbon – beside water, carbon is one of the most important requirements for microbial growth. It is the structural backbone of living matter and needed for all the organic compounds that make up living cells. Half of the dry-weight of a typical bacterium is carbon. Autotrophs are organisms that grow in inorganic media and receive their carbon from organic materials such as proteins, carbohydrates, and lipids. They get their carbon from carbon dioxide (CO2). Heterotrophs are organisms that can only grow in organic media, receive organic nutrients – primarily glucose. Nitrogen, Sulfur and Phosphorus – in addition to carbon, microorganisms need other elements in order to synthesize material. Protein synthesis requires large amounts of nitrogen as well as some Sulphur. DNA and RNA synthesis also requires nitrogen and some phosphorus, as does the synthesis of ATP (which is the molecule responsible for the storage and transfer of chemical energy within the cell). See text for more specific detail regarding specific applications of these elements. Trace Elements – microbes require very small amounts of other mineral elements such as iron, copper, molybdenum, and zinc – these are referred to as trace elements. Most are essential to the function of certain enzymes usually as cofactors. Sometimes added to lab media they are usually found in tap water. Vitamins – are a source of co-enzymes required for the formation of enzyme systems, particularly the B vitamins. Some do not require any vitamins Oxygen – many current forms of life require oxygen for aerobic respiration. Microbes that use or need oxygen (aerobes), extract more energy from nutrients than microbes that do not use or need oxygen (anaerobes). Refer to your text for more detailed definition of: obligate aerobes, facultative anaerobes, and those organisms harmed by oxygen: singlet oxygen, superoxide radicals, peroxide anions, hydroxyl radicals, aerotolerant anaerobes and microaerophiles Water – all cells require distilled water so that low molecular weight nutrients can cross the cell membrane Energy – needed for active transport, biosynthesis and biodegradation of cells. Two bioenergetics types of microbes exist: A) Phototroph uses radiant energy as a source, and B) Chemotroph depends on oxidation (adding O2) of chemical compounds as a n energy source. Some microbes use organic molecules, such as  BIO 351 Pg.21 glucose and others use inorganic compounds such as H2S (hydrogens sulfide) and NaNO2 (sodium nitrite). II Physical Requirements for Growth: The 3 most important physical factors that influence the growth and survival of cells are temperature, pH and the gaseous environment, understanding of the roles they play is cell metabolism is essential. Temperature most microbes grow in temperatures that favor humans but others grow at extreme temperatures. We classify them into 3 major groups: Psychrophiles (cold loving) -5 to 20°C. – most grow at 0-5 C. Mesophiles (moderate-temperature loving) 20 to 45°C., optimal 20-30 for plants, and 35-40 C. for warm blooded hosts Thermophiles (heat loving) 45-60°C., Facultative 37 C., optimal 45-60 C. and Obligate thermophiles 60, also called: Hyper or Extreme Thermophiles (extreme heat loving) >60 to 120°C. NOTE: Effect of Temperature on Growth and cellular enzymes. With increasing temperature, enzyme activity increases and at lower temperature, enzyme inactivation occurs and cellular metabolism diminishes. At zero degrees’ biochemical reactions cease LAB 5 EXERCISE: Experiment is done in groups of 4. Each group is assigned one organism. Organisms being used are: Serratia marcescens, Bacillus stearothermophilius, E. coli and Staph aureus. Temperature Experiment: 1. Each group will label the plates with your group # (1, 2, 3 or 4), the organisms that you are plating and the 4 different temperature incubations listed below in step 3 2. Each group will aseptically inoculate 4 plates with their particular organism, using a sterile swab and a single serpentine line 3. Each of the 4 plates will be incubated at a different temperature: 4°C, 20°C, 37°C, and 60°C, (by the technician) Please make sure place the plates in the corresponding labeled storage bin, according to correct corresponding temperature! BIO 351 Pg.22 pH refers to the acidity or alkalinity of a solution – most bacteria grow best in a narrow pH, between 6.5 and 7.5 – very few bacteria grow at an acidic pH below about 4.0. There are, however some bacteria called acidophiles that are remarkably tolerant of acidity. Molds and yeast grow over a greater pH range than do bacteria and at lower pH like 5.0 to 6.0. Bacteria cultured in the lab sometimes produce their own acids that sometimes interfere with their own growth Gaseous Environment in most cells is atmospheric oxygen, which is necessary for the bio oxidative process of respiration. These cells are known as aerobic form of cells. This oxygen plays a vital role in ATP formation and the availability of energy in a useful form for cell activities. Other cell types, lack the enzyme systems for respiration in the presence of oxygen and therefore must use an anaerobic form of respiration or fermentation. III Osmotic Pressure microbes obtain almost all of their nutrients in solution from surrounding water. Thus, they require water for growth. They are composed of 80-90% water. High osmotic pressure removes this necessary water from the cells. When a cell is in a solution whose concentration of solute is higher than the cell, it is considered to be hypertonic to the cell. Here cellular water passes out of the cell causing plasmolysis or shrinkage of the cell’s cytoplasm. Some (extreme halophiles) adapt well to this environment. Salt increases the osmotic pressure and is used to preserve foods – by drawing water out of microbial cells and preventing their growth. Some add as much as 30% salt. More common are facultative halophiles which do not require high salt concentrations and are able to grow at concentrations up to 2%. Most organisms must be grown in a nearly all water medium, agar is about 1.5% salt. Extremely low osmotic pressure or hypotonic environments (such as in distilled water) can cause water to enter the cell and those organisms with weaker cell walls, may lyse or rupture by such treatment. Hypertonic Solution – has a higher osmotic pressure and higher solute concentration and therefore has a lower water concentration – water leaves the cell and they tend to shrink or “plasmolyze.” Hypotonic Solution – has a lower osmotic pressure and lower solute concentration and therefore has a higher water concentration. Water tends to enter the cell. Cells with weak cell walls such as gram-negative bacteria sometimes will lyse or rupture due to excessive water intake, but most bacterial cells can live in a hypotonic solution Isotonic Solution – has equal concentration of solutes and equal water concentration, and therefore there is no osmosis Example: normal saline (0.85% NaCl) LAB EXERCISE 5: Environmental Osmotic Pressure Experiment This experiment will be done in groups of four (4), Each group will receive: 1. One set of 5 plates: each plate contains a different salt concentration:0.85%, 5%, 10%, 15% and 20% BIO351 Pg. 23 2.Each group will label their plates with the group # (1, 2, 3 or 4) and the salt concentration for each plate (if not already labeled) 3. Each salt plate will be dividing in half (draw a line down the middle of the plate on the agar side of the plate to label it)- write “E. coli” on one side and “S. aureus” on the other side of each plate. 4. Using a sterile swab, aseptically inoculate both E. coli and S. aureus on each plate, using a single serpentine line, for the 5 different salt concentrations Physical and Chemical Agents Control of microorganisms is essential in home, business, industry and medical fields to prevent and treat diseases and to avoid or inhibit the spoilage of food. Chemical and physical agents do this. A microbicidal effect is an effect that kills microbes immediately A microstatic effect is an effect that inhibits reproduction and maintains a microbial population at a constant size. Chemical Methods of Control: 1. Antiseptics – chemical substances used on living tissue to kill bacteria 2. Disinfectants – chemical substances used on non-living materials/surfaces 3. Chemotherapeutic agents – chemical substances that inhibit growth in living tissues Physical Methods of Control 1. Cell wall injury a) lysis of the cell wall weakens cells – called protoplasts b) failed synthesis inhibition of cells – called proloplast 2. Cell membrane damage – also due to lysis causing immediate cell death 3. Alteration of colloidal state of cytoplasm – certain agents cause denaturation of cytoplasmic proteins = enzyme inactivation = cell death due to rupturing of the molecular bonds of proteins 4. Inactivation of cellular enzymes – either competitive or non-competitive: a) Competitive – when a natural substrate is forced to compete for the active site on the enzymes surface with a chemically similar molecule. This blocks the enzyme’s ability to create end products (Reversible) b) Non-competitive – from a physical agent such as “mercuric chloride (HgCl2)” which uncoils the protein molecule and make it biologically inactive (Irreversible) 5. Interference with structure and function of DNA – the DNA molecule is the control center of the cell and a target area for destruction or inhibition. Some agents have an affinity for DNA and cause breakage or distortion of the molecule, which equals interference with replication role in protein synthesis.  BIO351 Pg. 22 Vitamins – are a source of co-enzymes required for the formation of enzyme systems, particularly the B vitamins. Some do not require any vitamins Oxygen – many current forms of life require oxygen for aerobic respiration. Microbes that use or need oxygen (aerobes), extract more energy from nutrients than microbes that do not use or need oxygen (anaerobes). Refer to your text for more detailed definition of: obligate aerobes, facultative anaerobes, and those organisms harmed by oxygen: singlet oxygen, superoxide radicals, peroxide anions, hydroxyl radicals, aerotolerant anaerobes and microaerophiles Water – all cells require distilled water so that low molecular weight nutrients can cross the cell membrane Energy – needed for active transport, biosynthesis and biodegradation of cells. Two bioenergetics types of microbes exist: A) Phototroph uses radiant energy as a source, and B) Chemotroph depends on oxidation (adding O2) of chemical compounds as a n energy source. Some microbes use organic molecules, such as glucose and others use inorganic compounds such as H2S (hydrogens sulfide) and NaNO2 (sodium nitrite). Physical and Chemical Agents Control of microorganisms is essential in home, business, industry and medical fields to prevent and treat diseases and to avoid or inhibit the spoilage of food. Chemical and physical agents do this. A microbicidal effect is an effect that kills microbes immediately A microstatic effect is an effect that inhibits reproduction and maintains a microbial population at a constant size. Chemical Methods of Control: 1. Antiseptics – chemical substances used on living tissue to kill bacteria 2. Disinfectants – chemical substances used on non-living materials/surfaces 3. Chemotherapeutic agents – chemical substances that inhibit growth in living tissues Physical Methods of Control 1. Cell wall injury a) lysis of the cell wall weakens cells – called protoplasts b) failed synthesis inhibition of cells – called proloplast 2. Cell membrane damage – also due to lysis causing immediate cell death 3. Alteration of colloidal state of cytoplasm – certain agents cause denaturation of cytoplasmic proteins = enzyme inactivation = cell death due to rupturing of the molecular bonds of proteins BIO351 Pg.23 4. Inactivation of cellular enzymes – either competitive or non-competitive: a) Competitive – when a natural substrate is forced to compete for the active site on the enzymes surface with a chemically similar molecule. This blocks the enzyme’s ability to create end products (Reversible) b) Non-competitive – from a physical agent such as “mercuric chloride (HgCl2)” which uncoils the protein molecule and make it biologically inactive (Irreversible) 5. Interference with structure and function of DNA – the DNA molecule is the control center of the cell and a target area for destruction or inhibition. Some agents have an affinity for DNA and cause breakage or distortion of the molecule, which equals interference with replication role in protein synthesis LAB 6: Physical Agents of Control – Electromagnetic Radiation Some forms of electromagnetic radiation can have a lethal effect on cells and therefore can be used as a microbial control. Radiation that has enough energy to be microbicidal are the short-wave radiations. i.e. 300 nm and below. This includes UV, gamma and x-rays. The high-wave length radiations, above 300 nm do not have enough energy to destroy cells – See Fig.1 on Page 79 of Lab Manual. Gamma Radiation which is an ionizing form of radiation has a non-specific damaging effect on any molecule in its path and will undergo ionization -where cells will lose their chemical structure and activity. U-V Light which has a lower content has a lethal effect with 210-300 nm which is absorbed by nucleic acids. The DNA is the primary site of damage. The major effect is “thymine dimerization” which is the covalent bonding of 2 adjacent thymine molecules on one strand of nucleic acid in the DNA molecule. It distorts the configuration of the DNA molecule which interferes with DNA replication and transcription during protein synthesis. Gamma and X-ray are ionizing forms of radiation that can transfer their energy through “quanta” (photons) to the matter through which they pass – this causes excitation and loss of electrons in their path. LAB 6 – Lab Exercise for Effect of Temperature on Bacterial Growth Based on observation of their incubated plates, students will score the growth of each organism at the 4 different temperatures: 4 C., 20 C., 37 C. and 60 C. as: 0 = no growth, 1+ = scant growth, 2+ = moderate growth and 3+ = abundant growth. They will further classify the organism as a Psychrophile, Mesophile or Thermophile: Temperature Serratia Bacillus E. coli Staphylococcus marcescens stearothermophilius aureus 4 C. (refrig) 20 C. RT 37 C.(body) 60 C. Classification BIO351 Pg.24 LAB 6 – Lab Exercise for Osmotic Pressure Students will examine their 5 plates with varied % of salt in the medium and compare the results of 0.85%, 5%, 10%, 15% and 25% and grade the bacterial growth of each of the plated organisms as follows: 0 = no growth, 1+ = scant growth, 2+ = moderate growth and 3+ = abundant growth. They will then describe the range of growth and identify the optimal NaCl concentration for each. Indicate optimal growth: Organism 0.85% 5% 10% 15% 25% E. coli S. aureus Optimal: LAB 6 and 7: Standard Qualitative Analysis of Water – Updated 11/2019 (AP) General Microbiology of Water: the importance of drinking water supplies cannot be overemphasized. With increased industrialization, water sources for consumption and recreation have become adulterated with industrial as well as animal and human waste. As a result, water has become a formidable factor in disease transmission. Polluted water containing organic matter becomes nutrients for growth and multiplication of microorganisms. Nonpathogenic organisms are not a concern but intestinal contaminants of fecal origin are responsible for intestinal infections such as: bacillary dysentery, typhoid fever, cholera and paratyphoid fever. The WHO (World Health Organization) estimates that 1.7 million deaths per year result from unsafe water supplies. Most of these from diahhreal diseases and 90% of the deaths occur in children living in developing countries where there are unsanitary conditions and water issues. The WHO estimates that another 3.4 million deaths annually are caused by dangerous water-borne enteric bacterial pathogens such as: Shigella dysenteriae, Campylobacter jejuni, Salmonella typhi and Vibrio cholera. In addition, there are about 200 million annual infections by numerous parasites and helminth diseases associated with water. The U.S. infection incidence is much lower than the rest of the world and occur sporadically. Water screening then, is important and is examined to detect E. coli, the bacterium that indicates fecal pollution. Since E. coli is always present in the human intestine, its presence in water alerts public health officials to the possible presence of other human or animal intestinal pathogens. There are both qualitative and quantitative methods used to determine the sanitary condition of water. PRINCIPLE: The 3 basic tests to detect coliform bacteria in water are the presumptive, confirmed and completed tests. The tests are performed sequentially on each sample under analysis. They detect the presence of coliform bacteria (indicators of fecal contamination). – the gram negative, non-spore forming bacilli that ferment lactose with the production of acid and gas that is detectable following 24-hour incubation period at 37 C. NOTE: We will be only doing the Presumptive and Confirmed tests – we do not do the Completed Test BIO351 Pg.25 Water Analysis: 1. The Presumptive Test is specific for detection of coliform bacteria. Measured aliquots of water to be tested are added to a lactose fermentation broth containing an inverted Durham gas vial. Because these bacteria are capable of using lactose as a carbon source (the other enteric organisms are not) their detection is facilitated by the use of this medium. Tubes of lactose medium are inoculated with 10-ml, 1-ml, and 0.1-ml aliquots of the water sample. The series has at least 3 groups, each composed of 5 tubes of the specified medium. The tubes in each group are then inoculated with the designated volume of water sample. The greater the number of tubes in the test, the greater the sensitivity of the test – See Lab One directions, page 111. Lab 6: Part A: The Presumptive Test: Students work in groups of 4: 1. Each group takes one unknown Sample 1, 2, 3, or 4 2. There will be 4 large test tube racks (one for each group): 5 Double Strength (2x) Lactose Broths and 10 Single Strength (1x) Lactose broths Pipette the unknown sample as per Procedure: Lab One – page 111 Unknown Sample 10-ml to 5 tubes of LB2x 1-ml to 5 tubes of LB1x 0.1-ml to 5 tubes of LB1x NOTE: Development of gas in any of the tubes is presumptive evidence of the presence of coliform bacteria in the sample. This test also allows one to get an idea of the number of coliform organisms’ present, by means of the “most probable number (MPN) test. The MPN is estimated by determining the number of tubes in each group that show gas following the incubation period. See Table 1 on page 112 - Lab Manual. Lab 7: Part B 1. Read MPN Test, 2. Set up Confirmed Test, 3. Set up Chemical Agent TSA Plate Inoculation test, 4. Set up Kirby-Bauer Plate Inoculation. The Confirmed Test – the presence of a positive or doubtful presumptive test immediately means that the water sample is nonpotable. Confirmation of these results is necessary because positive presumptive tests may be the result of organisms that are non-coliform in origin and that are not recognized as indicators of fecal pollution. The “Confirmed Test” requires that selective and differential media such as eosin- methylene blue (EMB) or Endo agar be streaked from a positive lactose broth tube obtained from the Presumptive Test. EMB contains the dye methylene blue, which inhibits the growth of gram-positive organisms. In the presence of an acid- environment, EMB forms a complex that precipitates out onto the coliform colonies, producing dark centers and a green-metallic sheen. The reaction is characteristic for E. coli, the major indicator of fecal pollution. Endo agar is a nutrient medium containing the dye fuchsin, which is present in a decolorized state. In the presence of acid produced by the coliform bacteria, fuchsin forms a dark pink complex that turns the E, coli colonies and the surrounding medium - pink NOTE: See “Confirmed Test” Procedure – Lab Two and Three – page 111-111 BIO351 Pg.26 WEEK 7 – PROCEDURE: The Confirmed Test: Page-110-111 Lab Manual 1. Label 3 EMB or Endo agar plates with the source of water sample (swage, pond and tap) 2. Using a positive-24-hour lactose broth culture, from the sewage water series from the presumptive test, streak the surface of one EMB or Endo agar plate, as described to obtain discrete colonies (4 – quadrant streak); 3. Repeat Step 2 from the positive lactose broth cultures from the pond and tap water series from the presumptive test to the remaining 2 plates 4. Incubate all plates in the inverted position for 24 hours at 37 C. WEEK 8 – PROCEDURE: Reading the Confirmed Test 1. Examine all the 3 plates from your confirmed test for the presence or absence of E. coli colonies (refer to the description of the confirmed test in the experiment introduction, “green-metallic sheen” – record the results in the Lab Report 2. Based on your results, determine whether each of the samples is potable or nonpotable. The presence of E. coli is a positive confirmed test. The absence of E. coli is a negative test, indicating that the water is not contaminated with fecal wastes and is therefore potable. Record the results in your Lab Report The Completed Test – we do not do this in Lab, however, the Completed Test is the final analysis of the water sample. It is used to examine the coliform colonies that appeared on the EMB or Endo agar plates used in the confirmed test. An isolated colony is picked up from the confirmatory test and inoculated into a tube of lactose broth and streaked on a nutrient agar slant to perform a Gram Stain. Following inoculation and incubation, tubes showing acid and gas in the lactose broth and presence of gram-negative bacteria, on microscopic examination are further confirmation of the presence of E-coli, and they are indicative of a positive completed test. WEEK 7 AND 8: MICROBIAL GROWTH CONTROL Chemical Methods of Control: 1. Antiseptics – chemical substances used on living tissue to kill bacteria 2. Disinfectants – chemical substances used on non-living materials/surfaces 3. Chemotherapeutic agents – chemical substances that inhibit growth in living BIO351 Page 27 WEEK 7 AND 8: MICROBIAL GROWTH CONTROL - Chemical Methods of Control: WEEK-7: Chemical Agents of Control Experiment–Disinfectants and Antiseptics Principle: This procedure requires heavy inoculation of an agar plate with the test organism Sterile, color-coded filter-paper discs are impregnated with a different antiseptic and equally spaced on the inoculated agar plate. Following incubation, the agar plate is examined (Week 8) for zones of inhibition (area of no microbial growth) surrounding the discs. A zone of inhibition is indicative of microbicidal activity against an organism. Absence of a zone f inhibition indicates that the chemical was ineffective against the test organism. NOTE: the size of the zone of inhibition is not indicative of the degree of effectiveness of the chemical agent. Antiseptic susceptibility is seen in Fig.1 – page 98 of Lab Manual. Week-7 Experiment 1: Students work in groups of 4: 4 TSA Plates per Group PROCEDURE: Control Experiment–Disinfectants and Antiseptics – Page 99 1. Students label their plates with their Group# and organism and 12, 3, 6 and 9 o’clock positions on the agar side of the plate. Students aseptically inoculate labeled TSA plates by streaking them with a sterile swab with both vertical and horizontal directions and around the edge 2. Color-coded Sensi-discs according to chemical agents: red=chlorine bleach, ____=tincture of iodine, ______=3% Hydrogen Peroxide (H2O2), and ______=70% Isopropyl Alcohol 3. Using forceps, dipped in alcohol and flamed, expose 4 discs of the same color into one of chemical solutions, i.e. Red discs=bleach, after immersing, drain the discs on absorbent paper, immediately prior to placing one disc on each of the 4 plates. Place each disc 2 cm in from the edge of the plate. Start with a red/bleach disc at 12 o’clock position on each plate. Gently press the disc down with the forceps so that they adhere to the surface of the agar 4. Impregnate the remaining discs as described in step 3. Place one of each remaining colored disc on the surface of each of the 3 remaining plates at 3 o’clock, 6 o’clock, 9 o’clock on each of the plates. 5. Place a plain unsoaked negative control paper disc in the center of each plate. 6. Incubate all plates in an inverted position for 24-28 hours at 37 C. Week-8 Results: Results of Chemical Agents of Control Experiment Students worked in groups of 4 and performed heavy swab inoculation of 4 organisms onto 4 TSA Plates that are then incubated at 37 C. for 48 hours The 4 organisms are: The 4 chemicals are 1. E. coli – gram neg 1. 5% Bleach – 12 o’clock 2. S. aureus – gram pos 2. Tincture of Iodine – 3 “ 3. Pseudomonas aeruginosa – gram neg 3. 3% Hydrogen Peroxide-6 “ 4. Bacillus cereus – gram pos 4. 70% Isopropyl Alcohol-9 “  (spore-forming organism BIO351 Page 28 Week 8 Lab Results: Chemical Agents of Control Experiment(cont) RESULTS: 1. Observe all the plates for the presence of a zone of inhibition surrounding each of the impregnated discs, indicating effectiveness that chemical is “microbicidal” against the organism, no zone of inhibition indicated lack of effectiveness. NOTE: The size of the zone of inhibition is not indicative of the degree of effectiveness of the chemical agent, where with antibiotics, it is. 2. Record your observations in Lab Report- Pg.101-102, chart both grids to see by organism in grid 1 and then by bacterial group (gm+/-) in grid 2 NOTE: Organisms vary in their susceptibility to chemicals. Bacterial spores are the most resistant forms. Capsulated bacteria are more resistant that non- capsulated; acid-fast and older cells (metabolically les active) are more resistant than younger cells. Awareness of this affects the choice of chemical that is best. See explanation of factors affecting efficiency of chemicals – pg.97-98 WEEK 7: Chemotherapeutic Agent of Control Experiment #2 – Page 79-88 Principle: The available chemotherapeutic agents very in their scope of antimicrobial activity. Some have a limited spectrum of activity being effective against only one group of microorganisms. Other, exhibit broad-spectrum activity against against a range of microorganisms. The drug susceptibility of many pathogens is known, but it is sometimes necessary to determine the drug of choice. A standardized diffusion procedure with filter-paper discs on agar, known as the Kirby- Bauer method, is frequently used to determine the drug susceptibility of microorganisms isolated from infectious processes. The method allows rapid determination of the efficacy of a drug by measuring the diameter of the zone of inhibition that results from diffusion of the agent into the medium surrounding the disc. In this procedure, filter-paper discs of uniform size are impregnated with specified concentrations of different antibiotics and then placed on the surface of an agar plate that has been seeded with the organism to be tested. The medium of choice is Mueller-Hinton agar, with a pH of 7.2 to 7.4 which is poured into plates to a uniform depth of 5 mm and refrigerated after solidification. Prior to use, the plates are transferred to an incubator at 37 C. for 10-20 minutes to dry off the moisture that develops on the agar surface. The plates are then heavily inoculated with a standardized inoculum by means of a cotton swab to ensure the confluent growth of the organism. The discs are aseptically applied to the surface of the agar at well-spaced intervals. Once applied, each disc is gently touched with a sterile applicator stick to ensure its firm contact with the agar surface. Following incubation, the plates are examined (Week 8) for the presence of growth inhibition, which is indicated by a clear zone of inhibition, which is affected by other variables such as: 1. The ability and rate of diffusion of the antibiotic into the medium and its interaction with the test organism  2. The number of organisms inoculated 3. The growth rate of the organism BIO351 Page 29 A measurement of the diameter of the zone of inhibition, in millimeters is made, and its size is compared to that contained in a standardized chart, which is shown in Table 2 – “Zone Diameter Interpretive Standards for Organisms Other Than Haemophilus and Neisseria gonorrhoeae” – on Page 89 of Lab Manual. Based on this comparison, the test organism is determined to be resistant, or susceptible to the antibiotic.