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Streaking Methods

Streaking Methods

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Published by: chocoholic potchi on Jul 11, 2009
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01/06/2013

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Lab 5 – Isolation Techniques Ninoska Garcia-Ortiz 063 053 2Objective:• To understand the principle of bacterial isolation• To recognize the difference between the streak plate and the pour plate• To understand how to use the two quantitatively• To be able to write a description of some bacterial colonies• To be able to identify types in a mixtureTABLE 1 - Isolation by Streaking Results/DiscussionBACTERIA MORPHOLOGYStaphylococcus epidermis Round scappled, light beige, flatraised spreading edge.Escherichia coli Lobate, light beige, irregular, irregular andspreading.Bacillus subtilis Irregular spreading, irregular lobatePseudomonas seruginosa Transparent coloniesSerratia marcescens Round scappled, red, flatMix 1 Escherichia Coli (E.C)Bacillus subtilis (B.S)Mix 2 Serratia marcescens (S.M)TABLE 2 - Isolation by Pouring Results/DiscussionPLATE DESCRIPTION/OBSERVATION3Mix1 diluted X 103'sprinkles', blotches, round colonies, many growing into theagar 4Mix1 diluted X 104Growing into agar, irregular, round, sprinkles5Mix1 diluted X 105Growing into agar, irregular, round, sprinkles6Mix1 diluted X 106Relatively little growth, growing into agar , sprinkles7Mix1 diluted X 107Irregular, spreading, moderate growth, growing into agar,sprinklesMix 1 : (Staphylococcus epidermis (S.E), Escherichia Coli(E.C), Bacillus subtilis (B.S)Discussion:Isolation by streaking:- Table 1- Formation of colonies was successfully achieved in all seven plates.- In mix 1, only 2 of the 3 colonies were recognized based ontheir morphology:Escherichia Coli (E.C) & Bacillus subtilis (B.S).- In mix 2, only one of the three colonies was recognized based on their morphology:Serratia marcescens (S.M)- One possible explanation could be due to the originalnumber of microorganism in thesample.- The fact that some bacteria are more fastidious than otherscould be another explanation.- Eg. E. coli is non-fastidious so it can grow more easily thanothers.Isolation by pouring:- Table 2- Mix 1 was used for dilution of the pouring plates.- E. coli was the only bacteria recognized in the pour plate.- Absence of “sprinkles” on streak plates is the maindifference when compared to the pour  plates.- Microbes that were streaked on the surface of the agar platesgrew more freely as those thatwere mixed with the agar.Calculated concentration:- Plate 5 (from pour plates)- 43 colonies (counted)- 105 is the dilution factor - 10 is the 0.1 ml sample plated43 X 105 X10 = 43 000 000 bacteria/mlConclusion:- Lab objective was met.- Bacterial isolation was achieved; contributing tounderstanding of the principle- Experiment helped recognize the differences between streak  plates and pour plates- Quantitative form was achieved by applying the formula:o # of colonies on chosen plate multiplied by the dilutionfactor multiplied by 10- Plate descriptions were capable due to the examples of morphology- Control plates allowed for identification of type of bacteriain mixture.
 
Aseptic techniques must be used to reduce thelikelihood of bacterial contamination. This usuallyinvolves disinfection of working areas, minimising possible access by bacteria from the air to exposedmedia, and use of flames to kill bacteria whichmight enter vessels as they are opened.
Bacterial growthBacterial growth refers to an increase in cell numbers rather than an increase in cell size. The process by which bacterialcells divide to reproduce themselves is known as binarytransverse fission. The time taken from cell formation to celldivision is called the generation time. The generation time cantherefore be defined as the time taken for the cell count todouble.The curve shown in Figure 9 shows the phases of bacterialgrowth following inoculation of bacteria into a new growthmedium. The following phases can be identified:1. Lag phase: There is usually some delay in growthfollowing inoculation of bacteria into a new medium, duringwhich time the bacteria adapt to the medium and synthesisethe enzymes needed to break down the substances in thegrowth medium.2. Log phase: Once the bacteria have adapted to the newmedium they start to reproduce quickly and their numbersmultiply evenly for each increment of time. A plot of the lognumber of cells against time gives a linear relationship: this istherefore called the log phase. The cells are at their greatestactivity in this phase. Transferring cultures to a fresh mediumat regular intervals can maintain the cells in an active state. Anactive culture can rapidly dominate any new environment.3. Stationary phase: As the bacteria dominate the growthmedium, they deplete the available nutrients or toxic waste products accumulate, slowing the rate of reproduction. At thesame time, cells are dying off: A state of equilibrium isreached between the death of old cells and formation of newcells, resulting in no net change in cell numbers. This phase iscalled the stationary phase.4. Death phase: In the next phase the formation of new cellsceases and the existing cells gradually die off: This is calledthe death phase.5. The log phase can be prolonged by removing toxic waste, by adding more nutrients or both.The log phase can be prolonged by removing toxic waste, byadding more nutrients or both.The log phase can be prolonged by removing toxic waste, byadding more nutrients or both.Figure 9. The four phases of bacterial growth.Factors affecting bacterial growthBacterial growth is affected by (1) temperature, (2) nutrientavailability, (3) water supply, (4) oxygen supply, and (5)acidity of the medium.Temperature: Theoretically, bacteria can grow at alltemperatures between the freezing point of water and thetemperature at which protein or protoplasm coagulates.Somewhere between these maximum and minimum points liesthe optimum temperature at which the bacteria grow best.Temperatures below the minimum stop bacterial growth butdo not kill the organism. However, if the temperature is raisedabove the maximum, bacteria are soon killed. Most cells dieafter exposure to heat treatments in the order of 70°C for 15seconds, although spore-forming organisms require moresevere heat treatment, e.g. live steam at 120°C for 30 minutes.

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