Question

Can you write about the function of the following reagents in the clonogenic assay.

1. Agar
2. Crystal violet
3. PBS

Thank you in advance, I would appreciate it a lot!!!

Answer 1

Agar:

agar is a jelly like substance having two components,the linear polysaccharide agarose, and a heterogeneous mixture of smaller molecules called agaropectin.

Agar can be used as a laxative, an appetite suppressant, a vegetarian substitute for gelatin, a thickener for soups, in fruit preserves, ice cream, and other desserts, as a clarifying agent in brewing, and for sizing paper and fabrics.

In microbiology,Agar is a complex gelatinous carbohydrate, and is added to the LB broth (Lysogeny broth, Luria broth, Lennox broth, or Luria-Bertani medium), in order to form gel for bacteria to grow upon.It is typically sold commercially as a powder that can be mixed with water and prepared similarly to gelatin before use as a growth medium

The pH of agar reagent is 5-8 (50 °C, 1.5% in H2O, 100 °C/15 min.)

Crystal violet:

Crystal violet is also called as gentian violet or methyl violet 10B or hexamethyl pararosaniline chloride.It is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria.

Chemical formula: C25H30ClN3

The Crystal violet stain is a basic substance, and Congo red stain is an acidic substance. During the lab you are able to compare the colors to the ph scale, allowing it to show you that red substances end up being a 4 on the scale, and the violet substances are a 10 on the scale.Crystal violet can be used to kill or slow the growth of bacteria. It can also be used to destroy fungi by inhibiting their reproduction or ability to grow.

It can be used as a pH indicator (yellow to violet with the transition at a pH = 1.6) In the medical community, it is the active ingredient in Gram's Stain, used to classify bacteria.Gram-positive cells stain purple because they retain the crystal violet-iodine complex in their cell walls. Also because the crystal violet is a positive charge dye which is attracted to the negative charge in the cell and also the thicker peplidoglycan layer helps retain the dye more.

PBS:-

     PBS is Phosphate-buffered saline. It is used in biological research. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH.Micro organisms such as Cyanobacteria and single cell algae can grow in the PBS.