Simple stain 1

Lab  microbiology

The science of Microbiology is the study of microorganisms and their activities. These organisms are called Microorganisms because they require magnification to see it .
Rules of safety in microbiology Bacterial Staining Techniques

The student should wear a clean coat in all laboratory this is for protection from contamination with stainsµorganism. -Do not smoke or bring food to the laboratory do not put anything in your mouth which may have been in contact with the tables in the laboratory. -Clean your table top with disinfectant at the beginning of each laboratory period, do the same that after finishing. -Wash your hands with soap and water before leaving the laboratory.

-Many of microorganisms used in this laboratory may be pathogenic to human –so, certain rules are necessary to in sure your safety in the laboratory .-These rules are:

Preparing smear and simple stain

Bacteria are microscopic organisms. They are also colorless In order to study their structure, shape and other structural characteristics, it becomes necessary to make them more easily visible. Most of dyes used in microbiology may be classified chemically as a salt because it contains positively and negatively charged ions -basic dye is positively charged and have affinity for negative ions. Such as crystal violate, safranine , methylene blue -Acidic dye is negative charged have affinity for positive ions Such as eosin,nigrosin


Simple staining – there are two methods: 1- positive staining - where the actual cells are themselves colored and appear in a clear background; 2-negative staining – where the cells remain clear (uncolored) and the background is colored negative staining
positive staining

bacteria have affinity for basic dyes such as Crystal violate, Safranine and methylene blue because of the negative charges on cell wall acidic dyes such as acid fuchsin, eosine, nigrosin are repelled by the negative charges of bacteria

Purpose of staining

Study the morphology of bacteria Study the arrangement of cells Demonstrate the microorganism Identification of microorganism Study the special structure of microorganism



Bacteria can have several different shapes, but the primary shapes we will be observing are: Spherical or round cells – cocci Rod shaped – bacilliSpiral shaped – spirilla Some bacteria have characteristic arrangements Diplococci – divide in one plane and remain attached together after cell division.Streptococci – divide in one plane and form long chains of attached cells.Staphylococci – divide in many planes and remain attached together forming a “grape-like cluster

The major steps in preparing a stained microbial specimen for microscopically examination

Prepared a smear of specimen on glass slide A bacterial smear: is a dried preparation of bacterial cells on glass slide:- If the cells to be stained are from agar media) culture solid media) (place a small drop of water on the top of the slide using a heat sterilized inculcating loop and transfer microorganism from agar culture to the slide and mix them with the water spread the specimen over the slide.


One of the most errors in a smear preparation from agar culture is the use of too large inoculums If the cells are from broth culture transfer a drop of the broth to the slide without mixing with water spread the cell over the slide.

Fixation

Fix the smear to the slide by passing the slide through the flame of Bunsen burner. Most bacteria can be fixed to the slide and killed in this way without serious distortion of cell structure. Advantage of fixation Kill the microorganism . Adhere the cell on this slide. Fix or keep the cells in ordinary shape


Simple Stain:Is the application of a single stain to a fixed smear such as methylene blue, crystal violate, safranine
a single stain to create contrast between the bacteria and the back ground is referred to as simple stain .

Materials- 24 hour agar & broth culture of Staphylococcus epidermidis.-Microscope, Inoculating loop and needle, immersion oil, crystal violate, ' carbol fuchsin.
Procedure For the broth culture shake the culture tube and with the inoculating loop transfer 1 to 2 loop full of bacteria to the center of the slide, spread , when preparing a smear from plate, place a loop full of water in the center of the slide with the inoculating needle aseptically pick up a very small amount of culture and mix into the drop of water spread this out as above


Smear preparation

Result