NEGATIVE STAINING LAB PRACTICAL Flashcards
Why negative staining procedure?
Requires the use of an acidic stain
1. Used because heat fixation is not required and the cells are not subjected to distorting effects of chemical and heat
- Makes it possible to observe bacteria that are difficult to stain.
Procedure
- Place a drop of nigrosin at one end of the slide
- Place a loopful of the inoculum into the drop of the stain and mix with the loop
- Place a slide against the drop of suspended organisms at a 45 degree angle and allow the drop to spread along the edge of the applied slide
- Push the slide away from the previosuly spread drop of suspended organisms, forming a thin smear. Air dry the slide
Why gram stain procedure?
It divides bacterial cells into two major groups gram negative and gram positive —- which makes it important for differentiation and classification
Gram Stain procedure
- Obtain clean glass stains
- Using aseptic technique prepare a smear of an organism on the slide
- allow smears to air dry
- Gently flood smears with crystal violet and let it stand for one minute
- gently wash with tap water
- gently flood smears with gram’s iodine mordant and let stand for 1 minute
- gently wash with tap water
- decolorize with alchohol
- gently wash with tap water
- counterstain w safranin for 45 seconds
- gently wash with tap water
- blot dry with bibulous paper and examine under oil immersion
Who do acid fast stain procedure?
- Members of the Mycobacterium genus are visualized more clearly by acid fast because the waxy wall makes penetration by stains difficult
- because m tuberculosis and m leprae represent bacteria that are pathogenic to humans the stain is of diagnostic value in indentifying these organisms
Acid fast stain procedure
- Flood smear with carbol fuchsin and place over a beaker of water on a warm hot plate allowing the prepration to steam for 5minutes
- wash with tap water
- Decolorize with acid alchohol, adding the reagent drop by drop until the alchohol runs almost clear
- Wash with tap water
- Counterstain with methlene blue for 2 minutes
- wash smear with tap water
- blot dry with bilous paper and examine under oil immersion
Why so Spore Stain?
Members of the anaerobic genera Clostridium and Desulfotomaculum and the aerobic genus bacillis are spores
used to look at spores
Spore Procedure
- Flood smears with malachite green and place on top of a beaker of water sitting on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes
- Remove slides from hot plate, cool, and wash under running tap water
- Counterstain with safranin for 30 seconds
- Wash with tap water
- Blot dry with biulous paper and examine under oil immersion
Selective Media
- used to isolate specific groups of bacteria
- incorporate chemical substances that inhibit the growth of one type of bacteria while permitting the growth of another
- Phenylethyl alchohol agar
- Crystal violet agar
- 7.5% sodium chloride agar
Phenylethyl Alchohol Agar
used for the isolation of most gram positive organisms
-partially inhibitory to gram-negative organisms which may form visible colonies whose size and number are smaller than on other media
Crystal Violet Agar
selective for most gram negative microogranisms. Crystal violet dye exerts an inhibitory effect on most gram positive organisms
7.5% sodium chloride agar
inhibitory to most organisms other than halophilic microorganisms.
-most useful in the detection of members of the genus Staphylococcus
Differential/Selective Media
- Can distinguish among morphologically and biochemically related groups of organisms
- incorporate chemical compounds that following inoculation and incubation produce a characteristic change in the appearance of bacterial growth and or medium surrounding the colonies which permits differentation
Sometimes differential and selective media
somes they are combined in a single medium. MacConkeys agar is a good example because it contains bile salts and crystal violet which inhibit gram positive organisms and allow gram negative organisms to grow.
-MacConkeys agar contains substrate lactose and the pH indicator neutral red, which differentiates the re
What are the differential and selective medias?
- mannitol salt agar
- MacConkey’s Agar (Coliform bacilli) and (Dysentery, typhoid, and paratyphoid bacilli
- Eosin-methylene blue agar (levine)
Mannitol Salt Agar
- Contains a high salt concentration 7.5% NaCL which is inhibitory to the growth of most but not all bacteria other than the staphylocci
- performs a differential function too because it contains carbohydrate mannitol which some staphlococci are capable of fermenting and phenol red, a pH indicator for deteching acid producing by mannitol fermenting staphylococci
- fermenting staphylocci exhibit a yellow zone surrounding their growth and staphylocci that do not ferment mannitol will not produce a change in coloration
MacConkey agar
inhibitory action of crystal violet on the growth of gran positive organisms allows the isolation of gram negative bacteria
-incorporation of the carbohydrate lactose, bile salts, and the pH indicator neutral red permits differentiation of enteric bacteria on the basis on their ability to ferment lactose. The enteric bacteria are seperated in two groups (Colliform bacilli and Dysentery, typhoid, and paratyphoid bacilli)
Colliform Bacilli
- produce acid as a result of lactose fermentation
- the bacteria exhibit a red coloration on their surface
Dysentery, typhoid, and paratyphoid bacilli
are not lactose fermenters and therefore do not produce acid
-colonies appear tan and frequently transparent when grown in MacConkey agar
Eosin-methylene blue agar
permit differentiation between enteric lactose fermenters and nonfermenters and the identification of the colon bacillus e coli
-the e coli colonies are blue and black w a metallic green sheen caused by the large quantity of acid that is produced and that precipitates the dyes onto the growths surface
Enriched Media
media that have been suplemented with highly nutritious materials like blood, serum, yeast extract for the purpose of cultivating fastidious organisms
Blood Agar
is a type of enriched media
- blood incorporated into the medium is an enrichment ingredient for the cultivation of fastidious organisms such as streptococcus spp.
- the blood permits demonstration of the hemolytic properties of some MOs usually streptocci which hemolytic activites are classified as Gamma/Alpha/Beta Hemolysis
Gamma Hemolysis
-no lysis of red blood cells in result no significant change in the appearance of the medium surrounding the colonies
Alpha Hemolysis
Incomplete lysis of red blood cells, with reduction of hemoglobin to methemoglobin results in a greenish halo around the bacterial growth
beta Hemolysis
Lysis of red blood cells with complete destruction and use of hemoglobin by the orgnisms results in a clear zone surrounding the colonies
-produced by two types of betahemolysins streptolsin O and streptolysin S
Extracellular Enzymatic Activities of Microorangisms
Because nutrients like polysaccharides, lipids, and proteins cant break into the cell membrane they have to be hydrolyzed by specific extracellular enzymes into their respective basic building blocks so they can be transported into the cells and used for synthesis
Starch Hydrolysis
starch is a high molecular weight branching polymer composed of glucose molecules linked together by glycosidic bonds
- amalyse is use for the degradation of these macromolecule so it can be hydrolysized into dextrins then ultimately maltose molecules
- the final hydrolysis of this disaccharide is catalyzed by maltase and has a low molecular weight and soluble glucose molecules that can be transported into the cell and is used for energy
Starch Hydrolysis in this experiment
Starch agar is used to demonstrate the hydrolytic activities of these exoenzymes
the medium is composed of nutrient agar supplemented with starch that serves as a polysaccharide substrate
-the detection of hydrolytic activity followring the growth period is made by the starch test by performing the starch test to determine the presence or absence of starch in the medium
Results in starch Hydrolysis experiment
Starch in the presence of iodine will impart a blue-black color indicating the absence of starch splitting enzymes so NEGATIVE (Surrounding by iodine)
Hydrolysis on the left will show positive (the streak is surrounded by the shit)
Lipid Hydrolysis
Triglycerides are degraded by enzymes called lipases that cleave the ester bonds in this molecule by the addition of water to form the building blocks glycerol and fatty acids and can be used for ATP
Lipid Hydrolysis Experiment
tributyrin agar is used to demonstrate it. After innoculation organisms that excrete lipase will show a zone of lipolyis that is represented by a clear surrounding area around the bacterial growth
- the loss of opacity is the result of hydrolytic reaction that causes soluble glycerol and fatty acids and represent a positive reaction
- if it retains its opacity it is negative
