ELN Lab 10 5-30.docx

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Microbiology and Cell Science
MCB 3020L

Chloe Evetts (8990-4674) Cameron Jacobs Enumeration of Microbes – Growth Curve Purpose The purpose of this lab is to learn the principles of determining how many bacteria exist in a sample. This is a very important part of microbiology because in order to develop new drugs, it is crucial to understand the effect of the drug on the survival of the organism being tested. Knowing how many bacteria exist in a sample is used in many facets, and in this lab, we will perform experiments to learn about this concept. In this experiment, we will use the optical density readings to correctly calculate the number of bacteria present, as well as the growth rate of the sample. Hypothesis By the end of the lab we will be able to successfully be able to determine how many bacteria exist in a sample. The amount of bacteria will be dependent on different parameters, such as oxygen, temperature, pH, salinity, radiation, light, nutrients available, pressure and others. Some bacteria will do better in certain conditions than others. However, bacteria are highly adaptable, so what may harm them in one experiment may not harm them as much if the experiment is repeated. The growth rate should be able to be identified by the optical density readings. Procedure Experiment 1: Growth Curve MATERIALS PER ROW: • Culture plate of Escherichia coli • Your unknown • 10 ml Liquid medium (BHI) • 8-well strips (per row) • Eppendorf tubes PROCEDURE • Take 8-well strips per row and label the edge • Take an Eppendorf tube, close the cap label it with your seat number, open it and add 1 ml BHI, close the cap • Make a suspension of your unknown in the Eppendorf tube – take a sample from an isolated colony with your loop and mix it with BHI – vortex well (approximate OD600 of >= 1.0) • Add 10 l of your bacterial suspension to 190 l of liquid medium in the 8 well strip – remember your well number • Per row: add 10 l of dilute E. coli to 190 l of liquid medium (positive control) and leave one well with 190 l BHI (negative control) • Program the spectrophotometer to read OD 600every 10 minutes for 12 hours • Start the program to read your samples. Your data set will be available to you after incubation has finished Calculation of Generation Time When growing exponentially by binary fission, the increase in a bacterial population is by geometric progression. If we start with one cell, when it divides, there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in the third generation, and so on. The generation time is the time interval required for the cells (or population) to divide. G (generation time) = t(time, in minutes or hours)/n(number of generations) = t/n http://textbookofbacteriology.net/growth_3.html To compare the rate of growth between different cultures the steepest part of the growth curve (log growth) is used to determine GT GT=((t2-t1)*log(2))/(log(OD2)-log(OD1)) Note: the generation time (GT) is calculated only from the log phase data of the growth curve Results BHI-37 Hours 1 2 3 4 5 6 Mean Standard Dev 0 0.12 0.099 0.11 0.12 0.099 0.11 0.109666667 0.009395034 0.5 0.13 0.1 0.11 0.11 0.101 0.149 0.116666667 0.019158984 1 0.14 0.11 0.121 0.122 0.113 0.238 0.140666667 0.048816664 1.5 0.13 0.1 0.122 0.13 0.124 0.36 0.161 0.098116258 2 0.323 0.245 0.136 0.178 0.276 0.312 0.245 0.074704752 2.5 0.47 0.44 0.48 0.59 0.57 0.52 0.511666667 0.059132619 3.5
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