Microbiology Lab Differential Stains

Name and Course Section: Beth Wyand, Section 701

Title: Differential Staining

Purpose: The purpose of this lab is to learn and implement Gram’s stain techniques to differentiate between types of bacteria.  Also, the lab will help to learn the differences between Gram positive and Gram negative bacteria, as well as explore properties that distinguish a variety of microorganisms

Procedure:  First, prepare an E. coli culture according to instructions from the lab manual and allow to incubate for 24-48 hours prior to the start of the experiment.  Then, using the S. epidermis and L acidophilus cultures from previous labs, incubate the samples for 12-24 hours in preparation for the experiment.  Once the incubation time has elapsed, prepare a S cerevisiae culture according to the lab manual.  

        Once all of the cultures have been appropriately prepared, disinfect the work area.  Prepare 4 slides, labeling each with the appropriate culture.  Prepare a smear for each slide and heat fix each organism.  Place the first slide in the staining tray and flood the slide with crystal violet dye and allow to sit for 1 minute.  Drain the excess dye and rise with tap water. Flood the sample slide with iodine solution and allow to sit for 1 minute.  Again, rinse with water.  Decolorize the slide by adding acetone alcohol and rinse with water.  Flood the slide with safranin and allow to sit for 30-60 seconds.  Rinse again with water, and blow with paper towels.  

        Repeat the steps above with each of the 4 samples.  Once completed, observe each slide under the microscope and record results.  Retain the e coli culture and S epidermis stock culture for future labs, and disinfect both the slides and the work area appropriately.

Lab Questions:

1. What is a differential stain? How is it different from a simple stain?

1. A differential stain is the use of two or more stains to categorize cells into groups. A simple stain uses one dye and shows only structure and size. A differential stain uses two or more dyes and is used to show morphology and distinguish cell types.

1. What is the difference between Gram-positive and Gram-negative cell walls?

1. The gram-positive cell wall appears thick and consists of numerous interconnecting layers. Also, in in the cell wall of gram-positive bacteria are certain acids. The Gram-negative cell wall contains a much thinner section of peptidoglycan – only two or three layers thick. There is also an outerlayer to the cell walls.

1. What is the purpose of crystal violet in the Gram’s stain procedure?

1. The stain is used to differentiate bacteria into 2 groups of pink and purple colors with gram positive being purple and gram negative being pink in color.

1. What is the purpose of iodine in the Gram’s stain procedure? What is a mordant?

1. Iodine helps retain the stain by forming an insoluble crystal violet-iodine complex. Both Gram-positive and Gram-negative bacteria remain purple.  A mordant is a substance that increases the cell affinity for a stain.  

1. What is the purpose of acetone-alcohol in the procedure?

1. The acetone-alcohol complex washes out of the thinner peptidoglycan layer of Gram-negative bacteria which become decolorized and allows a Gram-positive sample to remain purple.

1. What is the purpose of safranin in the Gram’s stain procedure?

1. Safranin serves as a counter stain.  So the gram positive samples are unaffected, while the gram negative retains a pink stain.

1. Why do gram positive cells stain purple?

1. Gram positive cells stain purple because their peptotidoglycan layer is thick enough, meaning that all Gram positive bacteria will retain their stain.  And after a number of steps, the cell walls are dehydrated so they retain the color.

1. Why do gram negative cells stain pink?

1. Gram negative cells stain pink because they have a thin peptidoglycan wall, and they will not retain any of the purple stain from crystal violet.  The decolorizer used dissolves the higher lipid content of Gram negative bacteria and the primary stain is able to seep into the solvent, washing away the dye, leaving the Gram negative bacteria unstained.

1. Which of the organisms stained gram negative?

1. The E coli stained negative

1. Which of the organisms stained gram positive?

1. The s epidermis, l acidophilus, and s cervisiae stained positive.

Conclusion

        From this lab, I was able to learn how to use Gram’s stain techniques as well as determining the certain types of bacterias that were each Gram positive and Gram negative.  I also learned how to tell the difference between the two based on microscopic views and the reasons why a bacteria is Gram negative or Gram positive depending on their cell wall.  

Lab Pictures

[pic 1]gram stain slides