Lab 1:  Introduction to Microbial Techniques

 

This lab is intended to familiarise students with proper procedures for handling bacteria, including spectrophotometry, dilutions, plating, and aseptic technique.  It will also allow you to understand the relationship between optical density of bacterial cultures and viable cell counts.

 

Optical density (OD) is a widely used technique to estimate the total number of bacterial cells present in a broth culture.  A spectrophotometer is used to measure the amount of light that can be transferred through a liquid.  If microbial cells are present, they will scatter the light and reduce the amount that can be transferred through the solution.  The exact amount of OD produced by a culture will depend on the concentration of cells present, the species and strain of microbe present, the growth conditions used, and the wavelength of the light being transmitted.  Thus, the relationship between OD and cell concentrations will depend on the specific strain and growth conditions studied.  OD measurements will detect all cells present in a solution, whether they are viable or not.

 

Dilutions and spread plating or pour plating are common techniques used to discover the viable cell count for a microbial culture.  The viable cell count is a measure of the concentration of cells present in a culture that are alive and able to grow.  Viable cells can be detected by plating cultures on (spread plating) or in (pour plating) solid nutrient media plates.  Single cells plated on solid media that allow the cells to grow will start replicating and produce a visible colony of cells, all descended from the same ancestral cell.  Spread plates will have colonies on top of the agar, while pour plates will have colonies embedded in the agar.  Sequential 10-fold dilutions are used to reduce cell concentrations in solution to a point where isolated single cells can be spread out on a plate so that each colony is separate from the other colonies, and the total number of colonies can be counted accurately.  This colony count can then be multiplied by the total dilution of the original culture, to obtain the number of viable cells present in the original culture.

 

Preparation of pour plates requires top agar overlays (on top of regular nutrient agar plates).  Top agar preparations contain lower concentrations of agar (~5-7 g/L) than normal solutions used to prepare agar plates (15 g/L).  The low agar concentration allows bacterial colonies to expand within the media.  Top agar overlays are also used for culturing bacteriophage and doing the Ames test, so that the virus particles or chemicals can diffuse through the media.

 

Top agar overlays are prepared by adding bacteria to ~ 2-3 ml of liquid top agar at 45-50°C.  Overlays are gently mixed, and then poured on top of prewarmed nutrient agar plates.  (Top agar must be maintained at 45-50°C until immediately before use – higher temperatures will kill bacteria, and lower temperatures will allow agar to solidify prematurely.)

 

 

phage mixing:

 

Because top agar is a viscous solution, standard vortexing tends to introduce air bubbles into the media, which may be difficult to distinguish from bacterial colonies.  To reduce the number of air bubbles resulting from mixing, molten agar solutions should be mixed using an appropriate technique.  The exact technique to use will depend on the type of tube the samples are prepared in. 

 

A technique called “phage mixing” has been developed for samples in standard glass test tubes.  This is done by holding a tube with the cap in the palm of the hand, and the fingers firmly holding on to the tube.  The tube is held at a 45° angle, and rotated in a circle 4-6 times in 1-2 seconds.  (The tube must remain at 45° while being rotated – this will be demonstrated in class.)  This technique allows for a gentle but thorough mixing, with minimal bubbles being introduced. 

 

Materials and Methods

 

Materials (per group of 3-4)

 

1 exponentially growing 5 ml E. coli K12 culture in LB

(0.05 ml of an overnight culture inoculated 3 hrs previous to lab, grown at 37 ºC)

sterile LB (5 ml aliquot)

sterile saline (15 ml)

11 test tubes

10 LB plates (room temperature) – for spread plates

10 LB plates (prewarmed in 37°C incubator) – for pour plates

10 LB top agar overlays (3 ml each)

(melted in microwave, cooled to 50°C in water bath)

– do not remove from water bath until immediately before use

 

Protocol

 

Week 1:

 

1.         Label 10 room temperature LB plates and 10 LB plates from the 37°C incubator.  Properly labelled plates should have the name of your group, the date, and the strain, media, and dilution present on the plate.  Return the prewarmed plates to the 37°C incubator.  (Top agar will not set as fast on prewarmed LB plates – this will allow phage overlays to be spread more evenly.)

 

2.         Measure the cell concentration using a spectrophotometer.  This should be done by adding 0.1000 ml of bacterial culture to 0.900 ml LB in a clean test tube, mixing well, transferring to a cuvette, and then reading the OD600 nm value.  The actual OD600 nm of your culture will be 10 X the measured value.

 

3.         Prepare a series of 10 dilution blanks (each with a volume of 0.900 ml) using sterile saline.  Use proper aseptic technique to avoid contaminating cultures.  Label tubes so that sequential 10-fold dilutions can be distinguished. 

 

4.         Transfer 0.1000 ml of undiluted bacterial culture to the first dilution blank, using proper aseptic technique.  Mix thoroughly.

 

5.         Aseptically transfer 0.1000 ml of your 10-1 dilution to the next dilution blank, and mix as before. Repeat this process for the 10-2 dilution, 10-3 dilution, etc. until the 10-10 dilution has been prepared.

 

6.         Aseptically transfer 0.1000 ml of each of your dilutions to room temperature LB plates, and spread the solution evenly over the surface of the plate with a sterilized spreader rod.  (Read the warnings in the Aseptic Techniques handout before doing this.)

 

7.         From the same dilutions, use 0.1000 ml of each of your dilutions to prepare 10 pour plates, following the instructions in the Aseptic Techniques handout and the phage mixing instructions above.  (Leave the plates at 37 ºC until you are ready to use them.)

 

8.         Allow pour plates to cool until agar has set.  Place your plates, lid side down, on the indicated tray for incubation.  (Plates will be incubated overnight at 37 ºC, and then refrigerated until the next lab period.) 

 

9.         Dispose of all materials and equipment in the proper places, and wipe down your workspace with ethanol.  Wash your hands before leaving the lab.

 

Week 2:

 

Count the number of colonies on your plates.  If a plate has greater than 300 colonies, it can be reported as Too Numerous To Count (TNTC).  Use plates with 30-300 colonies to calculate the concentration of viable bacterial cells present in the original culture.  Be sure to include all the dilutions (including the volume plated) in your calculation, and report your results to the proper number of significant figures.

 

The 30-300 range gives the most accurate results for plate counts.  Plates with greater than 300 colonies become hard to count because of colony overlap, and plates with less than 30 colonies are subject to large random errors relative to the small sample size.  If you do not have any plates in the 30-300 range, select the plate(s) that is/are closest to this range, and use it/them to calculate cell concentration.  Cell concentration values calculated from plates outside the 30-300 range should be reported as Estimated Plate Counts (ESPC).   Plates with colony numbers significantly higher than 300 can be estimated by counting the number of colonies on a fraction of the plate, and then multiplying to estimate the number on the entire plate.

 

Report

 

A short format report (see handout) is expected for this lab.  The report should include the measured OD600 nm value, the calculated OD600 nm value in the original culture, a table showing plate counts, and a calculation of the concentration of viable cells (cells/ml) present in the original culture.  Calculate the ratio of OD600 nm to viable cell concentration for your culture.  Show samples of all calculations.