Study guide for Mid-Term Micro Lab
Study guide for Mid-Term Micro Lab 81382 - MICR 3050 - 001
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Microbiology Lab 3051 Fall 2015 MidTerm Laboratory Exam Study Guide Labs 1 5 Objectives 1 Know the parts of the microscope and their functions Eyepiece contains the ocular lens which provides a magnification power of 10X to 15X usually This is where you look through Nosepiece holds the objective lenses and can be rotated easily to change magnification Objective lenses usually there are three or four objective lenses on a microscope consisting of 4X 10X 40X and 100x magnification powers In order to obtain the total magnification of an image you need to multiply the eyepiece lens power by the objective lens power So if you couple a 10X eyepiece lens with a 40X objective lens the total magnification is of 10 X 40 400 times Stage clips hold the slide in place Stage it is a at platform that supports the slide being analyzed Diaphragm it controls the intensity and size of the cone light projected on the specimen As a rule of thumb the more transparent the specimen less light is required Light source it projects light upwards through the diaphragm slide and lenses Base supports the microscope Condenser lens it helps to focus the light onto the sample analyzed They are particularly helpful when coupled with the highest objective lens Arm supports the microscope when carried Coarse adjustment knob when the knob is turned the stage moves up or down in order to coarse adjust the focus Fine adjustment knob used ne adjust the focus Know how to use the microscope adjusting for proper Viewing getting an image in focus using oil immersion and how to take care of a microscope Turn the revolving turret 2 so that the lowest power objective lens eg 4X is clicked into position Place the microscope slide on the stage 6 and fasten it with the stage clips Look at the objective lens 3 and the stage from the side and turn the focus knob 4 so the stage moves upward Move it up as far as it will go without letting the objective touch the coverslip Look through the eyepiece 1 and move the focus knob until the image comes into focus Adjust the condenser 7 and light intensity for the greatest amount of light Move the microscope slide around until the sample is in the centre of the eld of view what you see Use the focus knob 4 to place the sample into focus and readjust the condenser 7 and light intensity for the clearest image with low power objectives you might need to reduce the light intensity or shut the condenser When you have a clear image of your sample with the lowest power objective you can change to the next objective lenses You might need to readjust the sample into focus andor readjust the condenser and light intensity If you cannot focus on your specimen repeat steps 3 through 5 with the higher power objective lens in place Do not let the objective lens touch the slide When finished lower the stage click the low power lens into position and remove the slide Describe aseptic technique and understand why it is important lst inoculating loop is heated until it is red hot 2nd Cap is removed from slant and mouth of the tube is heated 3rd the organism is picked up from the slant with inoculating loop 4th Mouth of the tube is amed and inoculating loop is not amed 5th slant culture is recapped and returned to the test rack 6th Tube of the sterile agar slant is uncapped and mouth is amed 7 th slant surface is streaked with un amed loop in serpentine manner 8th Tube mouth is amed recapped and incubated 9th loop is amed and returned to receptacle This technique is important for keeping sterile cultures Compare and contrast how to make a bacterial smear from liquid media and solid media Liquid media You may spread a loopful or two of liquid culture directly onto a clean slide there is no need to add water to the slide first Solid media Smears made from solid cultures are usually better than those made from broth The most common problem is the use of too much inoculum Remember to only ll a small edge of your loop with inoculum from the culture ALWAYS inoculate a dry sample into a loopful or two of water Explain the importance of heat fixing and airdrying specimens on slides Heat xing Take a cotton swab and rub the inside of your cheeks to collect epithelium cells Smear these cells on a microscopy slide Completely airdry the slide without applying heat This should not take long because there is not much liquid on the slide anyway If you heat the slide before it is completely dry then you end up boiling apart the cells The vapor pressure inside the cells will burst them Heat fix the dried slide by quickly pulling it through a Bunsen burner 2x but in a way that the cells do not touch the ame Pull it through the ame with the cells on top and the ame below The slide should be pretty hot but you should still be capable of holding it in the palm of your hand without burning yourself You should just be capable of holding the slide Too high a temperature and you destroy the cells on the slide and on your skin Too low a temperature and the cells will not stick to the glass slide Distinguish between acidic and basic dyes and when to use each A basic dye is a stain that is cationic positively charged and will therefore react with material that is negatively charged The cytoplasm of all bacterial cells have a slight negative charge when growing in a medium of near neutral pH and will therefore attract and bind with basic dyes Some examples of basic dyes are crystal violet safranin basic fuchsin and methylene blue Acid dyes have negatively charged chromophores and are repelled by the bacterial surface forming a deposit around the organism They stain the background and leave the microbe transparent When to use them 1 simple nonspecific and 2 differential specific Simple stains will react with all microbes in an identical fashion They are useful solely for increasing contrast so that morphology size and arrangement of organisms can be determined Differential stains give varying results depending on the organism being treated These results are often helpful in identifying the microbe Describe simple staining and what bacterial characteristics can be observed using this technique The simple stain can be used to determine cell shape size and arrangement True to its name the simple stain is a very simple staining procedure involving only one stain You may choose from methylene blue Gram safranin and Gram crystal violet Procedure Perform a bacterial smear as discussed in Figure 352 on page 150 of your lab manual 2 Saturate the smear with basic dye for approximately 1 minute You may use crystal violet safranin or methylene blue 3 Rinse the slide gently with water 4 Carefully blot dry with bibulous paper 5 Observe the slide under the microscope using proper microscope technique Recognize the different bacterial morphologies Gram Positive Gram Negative Staphylococcus aureus Escherichia coli Step 1 Crystal Violet Step 2 Gram39s Iodine Step 3 Decolorizer G CDC 2 Alcohol or Acetone a Step 4 Sahanin Red A Bacterial Morphology Shapes Bacillus 3 Coccobacillus 5 5me coccus bacullus Spirillum Vibrio Spirochete 9 Understand the distribution of bacteria in our world It can be found Virtually anywhere air soil water inon usect 10 Compare and contrast grampositive and gramnegative cells What colors they stain using the Gram stain and how cell wall structure determines how they stain Grampositive bacteria stain purple with Gram stain This is because they have a thick cell wall without an outer membrane Gram Positive Example cyanobactcria Plasma Membrane ea Pcriplasnu39c space Petxidoglycan Plasma Membrane X Periplasmic space PEptidoglycan Outer membrane Pomlysaccharide and protein Gram Negative Gramnegative bacteria stain red with Gram stain This is because they have a thin cell wall with an outer membrane Example Salmonella Gram negative lippopolysacchrarideslipid A endotoxin they also have porins Gram positive techoic acids help anchor the layers of peptidoglycan together 11 12 Know all of the steps and stains used in a Gram stain Know the purpose of each stain and how bacteria look at each step Know which stain is the primary stain counterstain mordant and decolorizing agent Prepare a Slide SmearzTransfer a drop of the suspended culture to be examined on a slide with an inoculation loop If the culture is to be taken from a Petri dish or a slant culture tube first add a drop or a few loopful of water on the slide and aseptically transfer a minute amount of a colony from the Petri dish Note that only a very small amount of culture is needed a visual detection of the culture on an inoculation loop already indicates that too much is taken If staining a clinical specimen smear a very thin layer onto the slide using a wooden stick Do not use a cotton swab if at all possible as the cotton fibers may appear as artefacts The smear should be thin enough to dry completely within a few seconds Stain does not penetrate thickly applied specimens making interpretation very difficult Spread the culture with an inoculation loop to an even thin film over a circle of 15 cm in diameter approximately the size of a dime Thus a typical slide can simultaneously accommodate 3 to 4 small smears if more than one culture is to be examined Airdry the culture and fix it or over a gentle ame while moving the slide in a circular fashion to avoid localized overheating The applied heat helps the cell adhesion on the glass slide to make possible the subsequent rinsing of the smear with water without a significant loss of the culture Heat can also be applied to facilitate drying the the smear However ring patterns can form if heating is not uniform eg taking the slide in and out of the ame Gram Staining Add crystal violet which is the primary stain stain over the fixed culture Let stand for 60 seconds Pour off the stain and gently rinse the excess stain with a stream of water from a faucet or a plastic water bottle Add the iodine mordant solution on the smear enough to cover the fixed culture Let stand 60 seconds Pour off the iodine solution and rinse the slide with running water Shake off the excess water from the surface Add a few drops of decolorizer so the solution trickles down the slide Rinse it off with water after 1020 seconds Counterstain with basic fuchsin solution for 40 to 60 seconds Wash off the solution with water Blot with bibulous paper to remove the excess water Alternatively the slide may shaken to remove most of the water and airdried Understand the purpose of a streak plate and how you would do one The purpose of the streak plate is to obtain isolated colonies from an inoculum by creating areas of increasing dilution on a single plate How to preform a streak plate Four Quadrant Streak Loosen the cap of the bottle containing the inoculum Hold an inoculation loop in your right hand Flame the loop and allow it to cool Lift the test tube containing the inoculum with your left hand Remove the cap cotton wool plug of the test tube with the little finger of your right hand Flame the neck of the test tube Insert the loop into the culture broth and withdraw At all times hold the loop as still as possible Flame the neck of the test tube again Replace the cap cotton wool plug of the test tube using the little finger of your right hand Place the test tube in a rack For a liquid culture dip the loop into the broth or for solid media lightly touch a colony with the loop 9 Partially lift the lid of the Petri dish containing the solid medium Place a loopful of the culture on the agar surface on the area 1 Flame the loop and cool it for 5 seconds by touching an unused part of the agar surface close to the periphery of the plate and then drag it rapidly several times across the surface of areal 11 Remove the loop and close the Petri dish PWPPPL 9 13 14 12 Re ame and cool the loop and turn the petri dish 90 C then touch the loop to a corner of the culture in areal and drag it several times across the agar in area 2 hitting the original streak a few times The loop should never enter area 1 again Remove the loop and close the Petri dish Re ame and cool the loop and again turn the dish 90 C anticlockwise streak area 3 in the same manner as area 2 hitting last area several times Remove the loop and close the Petri dish Flame the loop again turn the dish 90 C and then drag the culture from a corner of a area3 across area 4 contacting area 3 several times and drag out the culture as illustrated Using a wider streak Do not let the loop touch any of the previously streaked areas The flaming of the loop at the points indicated is to effect the dilution of the culture so that fewer organisms are streaked in each area resulting in the final desired separation Remove the loop and close the Petri dish Tape the plate closed and incubates the plate in an inverted position in an incubator for 2448 hours Flame the loop before putting it aside 13 14 15 16 17 18 19 Define pure culture and pure colony Pure culture A culture in which 1 strainclone is present Pure colony A culture of genetic clones of the bacteria Know the purpose of a gelatin stab how you conducted this test and what a positive reaction for protease production would look like The purpose is to see if the microbe can use the protein gelatin as a source of carbon and energy for growth Use of gelatin is accomplished by the enzyme protease How to conduct this test An inoculum from a pure culture is transferred aseptically to a sterile tube of nutrient gelatin The inoculated tube is incubated at 3537 C for 24 hours and the results are determined Because even undigested gelatin becomes liquid at temperatures modestly above the incubation temperature it is important to determine whether the gelatin has actually been digested by protease This is accomplished by placing the tube into a refrigerator for a few minutes exposing it to temperatures that cause undigested gelatin to resolidify If the gelatin has been digested the medium in the tube will fail to solidify after refrigeration If protease is present the liquid medium will fail to solidify upon refrigeration If it has solidified it will not ow when the tube is tilted This is an indication of a negative test If the medium flows when the tube is tilted the gelatin has been digested This is a positive result for the presence of the enzyme protease 15 16 Describe endospores their function their unique characteristics and which genera produce them Endospore An endospore is a dormant tough nonreproductive structure produced by a small number of bacteria from the Firmicute family The primary function of mostendospores is to ensure the survival of a bacterium through periods of environmental stress Characteristics The resilience of an endospore can be explained in part by its unique cellular structure The outer proteinaceous coat surrounding the spore provides much of the chemical and enzymatic resistance Beneath the coat resides a very thick layer of specialized peptidoglycan called the cortex Proper cortex formation is needed for dehydration of the spore core which aids in resistance to high temperature A germ cell wall resides under the cortex This layer of peptidoglycan will become the cell wall of the bacterium after the endospore germinates The inner membrane under the germ cell wall is a major permeability barrier against several potentially damaging chemicals The center of the endospore the core exists in a very dehydrated state and houses the cell39s DNA ribosomes and large amounts of dipicolinic acid This endospore specific chemical can comprise up to 10 of the spore39s dry weight and appears to play a role in maintaining spore dormancy Small acidsoluble proteins SASPs are also only found in endospores These proteins tightly bind and condense the DNA and are in part responsible for resistance to UV light and DNAdamaging chemicals Other speciesspecific structures and chemicals associated with endospores include stalks toxin crystals or an additional outer glycoprotein layer called the exosporium Coat Outer Membrane Cortex Germ Cell Wall Inner Membrane Endospores are dormant alternate life forms produced by the genusBacillus the genus Clostridium and a number other genera of bacteria including Desulfotomaculum Sporosarcina Sporolactobacillus Oscillospira and Thermoactinomyces Understand the SchaefferFulton endospore staining procedure know the primary stain and counterstain Why heat is used as a mordant and What endospore producers look like under the microscope after being stained Perform a bacterial smear of Bacillus or the organism you want to stain as discussed in Figure 3 52 on page 150 of your lab manual 2 Place a small piece of bibulous paper over the smear Saturate the paper with malachite green 3 Heat the slide gently over the Bunsen burner for 5 minutes Be sure to keep the bibulous paper saturated with malachite green primary stain during heating If the slide is steaming you re okay if it stops steaming add more malachite green 4 Remove the bibulous paper from the slide and rinse the slide gently with water Dispose of the used bibulous paper in the trash DO NOT leave the bibulous paper in the sink or drain 5 17 18 19 20 Counterstain with safranin for 2 minutes 6 Rinse the slide gently with water 7 Carefully blot the slide dry with bibulous paper 8 Observe the slide under the microscope using proper microscope technique Endospores will stain green Parent cells will stain red Water decolorizes the cell Heat is used as the mordant bc it forces the green into the cells Know What genera of bacteria are acidfast and What structure causes them to be acidfast The acidfast stain is a differential stain used to identify acidfast organisms such as members of the genus Mycobacterium All of these characteristics of the microbe are due to the very THICK LIPID LAYER in the cell wall As you might expect the Mycobacterium stain Grampositive because of the THICK cell wall Describe the acidfast stain primary stain decolorization counterstain and know how acidfast cells and nonacid fast cells appear after staining The primary stain used in acidfast staining carbolfuchsin is lipidsoluble and contains phenol which helps the stain penetrate the cell wall This is further assisted by the addition of heat The smear is then rinsed with a very strong decolorizer which strips the stain from all nonacidfast cells but does not permeate the cell wall of acidfast organisms The decolorized nonacidfast cells then take up the counterstain methylblue 1 Perform a bacterial smear as discussed in Figure 352 on page 150 of your lab manual 2 Place a small piece of bibulous paper over the smear and saturate the paper with carbolfuchsin 3 Heat the slide gently over the Bunsen burner for 5 minutes Be sure to keep the bibulous paper saturated with carbolfuchsin during heating If the slide is steaming you re okay if it stops steaming add more carbolfuchsin 4 Remove the bibulous paper from the slide and rinse the slide gently with water Dispose of the used bibulous paper in the trash DO NOT leave the bibulous paper in the sink or drain 5 Decolorize the slide with acidalcohol until the rinsate runs clear 6 Rinse the slide gently with water 7 Counterstain with methylene blue for 2 minutes 8 Rinse the slide gently with water 9 Carefully blot the slide dry with bibulous paper 10 Observe the slide under the microscope using proper microscope technique Acidfast cells will stain fuchsia Nonacidfast cells will stain blue Describe bacterial capsules and their functions The capsule stain employs an acidic stain and a basic stain to detect capsule production Capsules are formed by organisms such as Klebsiella pneumonia Capsules protect bacteria from the phagocytic action of leukocytes and allow pathogens to invade the body If a pathogen loses its ability to form capsules it can become avirulent Bacterial capsules are nonionic so neither acidic nor basic stains will adhere to their surfaces Therefore the best way to visualize them is to stain the background using an acidic stain and to stain the cell itself using a basic stain Describe the use of negative staining to Visualize capsules and know how capsules appear after using this method Negative staining is an excellent way to determine an organism s cellular morphology Since the cells themselves are not stained their morphology is not distorted in any way The nigrosin provides a dark background against which the shapes of the unstained cells are clearly visible This method provides a high degree of contrast not available in most other staining procedures 1 Place a single drop of nigrosin on a clean microscope slide adjacent to the frosted edge 2 Using a amed loop and sterile technique remove some organism from your tube or plate and mix it into the drop of nigrosin Be sure there are no large clumps of organism but try to avoid spreading the drop 3 Place the end of another clean microscope slide at an angle to the end of the slide containing the organism and spread the drop out into a film This is done by contacting the drop of nigrosin with the clean microscope slide and using the capillary action of the dyemicroscope 21 22 23 slide to spread the nigrosin across the smear 4 Allow the film to air dry 5 Observe the slide under the microscope using proper microscope technique The background will be dark The bacterial cells will be stained purple The capsule if present will appear clear against the dark background background capsule bacterium Understand the methods we used to see motility and how motility or lack of was determined Wet mount look for directional movement different directions Hanging drop focus near the edge of the drop shallow field Stab technique wire and gelatin if the organisms are motile they will swim away from the line of inoculation Of the bacteria we used for the motility tests be able to name which bacteria are motile and nonmotile P vulgaris motile M luteus nonmotile For each of the following tests know the purpose what a positive reaction would look like what media and or reagents were used and how the media andor reagents work to show negative and positive reactions ex methyl red in MRVP turns red when the pH drops to 5 or less indicating mixed acid fermentation Sugar Fermentation Nitrate Reduction Mixed Acid Fermentation MR Starch Hydrolysis Butanediol Fermentation VP Casein Hydrolysis Citrate Test Tryptophan Degradation Oxidase Test Urea Hydrolysis Catalase Test SIM in place of TSI Sugar Fermentation Purpose Triple sugar agar is a differential media that is used to detect fermentation of sucrose lactose and glucose as well as the production of hydrogen sulfidegas This test is usually used to help identify gram negative rods The organism that only ferments glucose will be acid turning the entire medium yellow and raising the pH due to the breakdown of glucose and towards the end of 24 hours the tube will demonstrate a red slantampyellow butt to indicate fermentation of glucose alone TSI is the medium used If there is yellow color and gas then it had a full fermentation For example if the medium changes to yellow like it did with E coli then it is a fermenter If you observe a red color like we did with P aeruginosa then it did not ferment anything the pH is still unchanged from the control Summary of sugar fermentation yellow bubbles fermentation positive test Reddish color with no bubbles no sugar fermentation negative test S sonnei P aerugmos ninoculated E coli Here is a picture to visualize A3 Mixed Acid Fermentation Purpose This test determines whether the microbe performs mixed acids fermentation when supplied glucose Mixed acids fermentation results in accumulation of a variety of acids and a significant drop in the pH of the medium If the microbes perform mixed acid fermentation the medium will acquire an acidic pH and change color The medium used is MRVP broth Reagants added methyl red 5 drops If there is a color change from yellow to red then there is mixed acid fermentation If there is no change from the color red there is no mixed acid fermentation Summary Negative test9 E faecalis no color change Positive test9Ecoli changed from yellow to red indicating mixed acid fermentation 23 Butanediol fermentation VogesProskauer Test Purpose This test determines whether the microbe produces 23butanediol as a fermentation product from glucose The same medium as the MR test is used MRVP Reagants used 18 drops of Barrits A lightly shaken then 18 drops of Barrits B lightly shaken Positive test9 Eaerogenes color change to red Negative test9 E coli no color change Citrate Test Purpose The purpose is to see if the microbe can use the compound citrate as its sole source of carbon and energy for growth Medium Citrate agar slant streak and stab method Reagants none Positive test9 B subtilis growth on the slant and a change from green to blue indicated a positive test for the growth of citrate Negative test9 E coli no color change stays green Oxidase test Purpose A vital part of a bacterium s ability to generate ATP is a group of membranebound proteins called cytochromes Their function is to take the energetic electrons removed during metabolism of foods and transfer them to oxygen during oxidative metabolism The last of the cytochromes is called terminal oxidase This test is used to determine whether the oxidase cytochrome is present and functional Medium TSA plate Reagent Oxidase reagent Positive test9 Paeruginosa a deep indigo blue color will develop Negative test9 E coli no indigo Catalase Purpose The purpose is to see if the microbe has catalase a protective enzyme capable of destroying the dangerous chemical hydrogen peroxide Medium Nutrient agar slant Reagant Hydrogen peroxide Positive test9 S aureus Copious bubbles liberated in the hydrogen peroxide indicated presence of catalase Negative test9 E faecalis no bubbles Nitrate Reduction Purpose This test determines whether the microbe produces the enzymes nitrate reductase and nitrite reductase The two enzymes catalyze two reactions involved in converting starting compound nitrate into end product nitrogen gas How are the presence of these enzymes determined If a bacterium producing nitrate reductase is grown in a medium containing nitrate the enzyme converts the nitrate to nitrite Nitrite reacts with certain chemicals to yield a redcolored product If the bacterium also produces nitrite reductase nitrogen gas will be liberated Bubbles collecting in an inverted Durham tube indicate that nitrogen has been produced Medium Nitrate reduction broth with Durham tube Reagents Nitrate A Nitrate B if those don t work and the test is negative add a little zinc Positive test9 Ecoli A positive test for both enzymes consists of a turbid cloudy broth with pronounced gas bubbles trapped in the Durham tube If results like this are not observed testing for the individual enzymes can be done through addition of reagents with a positive test indicated by the broth turning red Negative test9E aerogenes no color change Starch hydrolysis Purpose The purpose is to see if the microbe can use starch a complex carbohydrate made from glucose as a source of carbon and energy for growth Use of starch is accomplished by an enzyme called alphaamylase Medium Starch Plate Reagents Iodine Positive test B subtilis After inoculation and overnight incubation iodine reagent is added to detect the presence of starch Iodine reagent complexes with starch to form a blueblack color in the culture medium Clear halos surrounding colonies is indicative of their ability to digest the starch in the medium due to the presence of alphaamylase Negative test9E coli no clear halos Casein Purpose To determine if an organism can produce the exoenzyme casesase Casein is a large protein that is responsible for the white color of milk This test is conducted on milk agar which is a complex media containing casein peptone and beef extract If an organism can produce casein then there will be a zone of clearing around the bacterial growth Medium Skim milk plate Reagents none Positive test B subtilis Clear zone around the bacteria Negative test9 E coli Tryptophan Degredation Purpose The indole test screens for the ability of an organism to degrade the amino acid tryptophan and produce indole Medium SIM agar Reagents 1012 drops of Kovac s reagent to detect tryptophan degradation Positive test9 P vulgaris Red band 24 25 26 Negative test9 B subtilis yellow Urea Hydrolysis Purpose The purpose is to see if the microbe can use the compound w as a source of carbon and energy for growth Use of w is accomplished by the enzyme urease Medium Urea agar Reagents none leave cap loose Positive test9 P vulgaris Bright pink color Negative test9 E coli red H2S production SIM instead of tryptone broth Purpose This test determines whether the microbe reduces sul trcontaining compounds to sulfides during the process of metabolism Medium SIM Reagents none Positive test9 Pvulgaris Black color please note If regions of the agar have been turned black the organism is producing hydrogen sulfide Other unrelated results can also be seen Cracks in the agar indicate gas production from the fermentation of sugars If the red agar has been turned yellow acids are being produced from at least one of the sugars contained within Negative test9 B subtilis no black Know the functional type mechanism of action What type of colonies will grow and how they can be distinguished on the following media MacConkey Agar MAC Phenylethyl Alcohol Agar PEA and Blood Agar MAC functional type selective and differential Mechanism of action selects for gram Lactose fermenters pink coloniesnonlactose fermenters will grow yellowclear phenylethyl alcohol agar PEA functional type selective Mechanism of action it selects for gram Gram will grow on the plate while gram will NOT grow on the plate Blood Agar functional type differential and enriched Mechanism of action detects hemolytic activity usually gram 9Clear zone betahemolysis pathogenic green zone alphahemolysis partially hemolyzed and no change gamma hemolysis or no hemolysis Understand how to construct a bacterial growth curve by measuring the turbidity of a broth culture Indirect method uses spectrophotometric measurements of developing turbity in a bacterial culture taken at different intervals These samples serve as an indeX of increasing cell mass For example take 2 points 2 and 4 on the absorbance scale and that represents doubling turbity From the 2 points draw the curve and perpendicular lines to the time intervals then the generation time can be calculated tA of 04 tA of 02 9 90 min 60 min After you find the growth rate g and k you can get the doubling time g 1k Direct method A specific volume of the bacterial suspension is placed on a microscope slide with a special grid The stain is added to visualize the bacteria and the cells are counted and multiplies by a factor to get the concentration Use this formula generation time 301t log10Nt log10N0 After this you can find the mean growth rate constant k Know the four phases of bacterial growth in a batch culture 1 Lag phase When a microorganism is introduced into the fresh medium it takes some time to adjust with the new environment This phase is termed as Lag phase in which cellular metabolism is accelerated cells are increasing in size but the bacteria are not able to replicate and therefore no increase in cell mass The length of the lag phase depends directly on the previous growth condition of the organism When the microorganism growing in a rich medium is inoculated into nutritionally poor medium the organism will take more time to adapt with the new environment The organism will start synthesising the necessary proteins coenzymes and vitamins needed for their growth and hence there will be a subsequent increase in the lag phase Similarly when an organism from a nutritionally poor medium is added to a nutritionally rich medium the organism can easily adapt to the environment it can start the cell division without any delay and therefore will have less lag phase it may be absent 2 Exponential or Logarithmic log phase During this phase the microorganisms are in a rapidly growing and dividing state Their metabolic activity increases and the organism begin the DNA replication by binary fission at a constant rate The growth medium is exploited at the maximal rate the culture reaches the maximum growth rate and the number of bacteria increases logarithmically exponentially and finally the single cell divide into two which replicate into four eight sixteen thirty two and so on That is 20 21 22 23 quot2 n is the number of generations This will result in a balanced growth The time taken by the bacteria to double in number during a specified time period is known as the generation time The generation time tends to vary with different organisms E coli divides in every 20 minutes hence its generation time is 20 minutes and for Staphylococcus aureus it is 30 minutes 3 Stationary phase As the bacterial population continues to grow all the nutrients in the growth medium are used up by the microorganism for their rapid multiplication This result in the accumulation of waste materials toxic metabolites and inhibitory compounds such as antibiotics in the medium This shifts the conditions of the medium such as pH and temperature thereby creating an unfavourable environment for the bacterial growth The reproduction rate will slow down the cells undergoing division is equal to the number of cell death and finally bacterium stops its division completely The cell number is not increased and thus the growth rate is stabilised If a cell taken from the stationary phase is introduced into a fresh medium the cell can easily move on the exponential phase and is able to perform its metabolic activities as usual 4 Decline or Death phase The depletion of nutrients and the subsequent accumulation of metabolic waste products and other toxic materials in the media will facilitates the bacterium to move on to the Death phase During this the bacterium completely loses its ability to reproduce Individual bacteria begin to die due to the unfavourable conditions and the death is rapid and at uniform rate The number of dead cells exceeds the number of live cells Some organisms which can resist this condition can survive in the environment by producing endospores lt Stationary phase gt 7 Death phase 3 Log I 5 exponential 3 phase 0 E 3 Z Lag phase 27 Be able to classify organisms based on their oxygen requirements obligate aerobes obligate anaerobes facultative anaerobes microaerophiles aerotolerant as shown by growth in uid thioglycollate medium FTM Obligate aerobes 02 requirement NEED oxygen Obligate anaerobes live in the absence of oxygen Facultative anaerobes can live in the absence or presence of 02 Microaerophiles These need oxygen because they cannot ferment or respire anaerobically However they are poisoned by high concentrations of oxygen They gather in the upper part of the test tube but not the very top Aerotolerant Organisms do not require oxygen as they metabolize energy anaerobically Define neutrophile acidophile and alkaliphile Neutrophile Any organism that thrives in a relatively neutral pH 7 Acidophilean organism that lives and thrives under acidic conditions a form of extremophile pH 2 or below Alkaiphile any organism that lives and thrives in an alkaline environment such as a soda lake a form of extremophile pH 911 Define AW hypotonic hypertonic plasmolysis halophiles halotolerant osmophiles Aw water activityamount of water available for a microorganism Hypotonic any solution that has a lower osmotic pressure than another solution In the biological fields this generally refers to a solution that has less solute and more water than another solution Hypertonic having a higher osmotic pressure than a particular uid typically a body uid or intracellular uid Plasmolysis The process in which cells lose water in a hypertonic solution 28 29 Halophiles organism that tolerates saline conditions Halotolerant adaptation of living organisms to conditions of high salinity Osmophiles bacteria that withstand high osmotic pressures Understand what happens to a cell when it is placed in a hypotonic or hypertonic environment Hypotonic If concentrations of dissolved solutes are less outside the cell than inside the concentration of water outside is correspondingly greater When a cell is exposed to such hypotonic conditions there is net water movement into the cell Cells without walls will swell and may burst lyse if excess water is not removed from the cell Cells with walls often benefit from the turgor pressure that develops in hypotonic environments Hypertonic If concentrations of dissolved solutes are greater outside the cell the concentration of water outside is correspondingly lower As a result water inside the cell will ow outwards to attain equilibrium causing the cell to shrink Know which organism that we tested should have been able to grow at the highest salt concentration Halophiles S aureus H salinarium The lab midterm exam will include the following types of questions multiple choice truefalse matching short answer fillin the blank with word bank and practicaltype questions in which you will have to look at something such as a slide plate tube etc and answer a question about it
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