NEGATIVE STAINING LAB PRACTICAL

Question 1

Why negative staining procedure?

Answer 1

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

2. Makes it possible to observe bacteria that are difficult to stain.

Question 2

Procedure

Answer 2

1. Place a drop of nigrosin at one end of the slide
2. Place a loopful of the inoculum into the drop of the stain and mix with the loop
3. 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
4. Push the slide away from the previosuly spread drop of suspended organisms, forming a thin smear. Air dry the slide

Question 3

Why gram stain procedure?

Answer 3

It divides bacterial cells into two major groups gram negative and gram positive —- which makes it important for differentiation and classification

Question 4

Gram Stain procedure

Answer 4

1. Obtain clean glass stains
2. Using aseptic technique prepare a smear of an organism on the slide
3. 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

Question 5

Who do acid fast stain procedure?

Answer 5

• 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

Question 6

Acid fast stain procedure

Answer 6

1. Flood smear with carbol fuchsin and place over a beaker of water on a warm hot plate allowing the prepration to steam for 5minutes
2. wash with tap water
3. Decolorize with acid alchohol, adding the reagent drop by drop until the alchohol runs almost clear
4. Wash with tap water
5. Counterstain with methlene blue for 2 minutes
6. wash smear with tap water
7. blot dry with bilous paper and examine under oil immersion

Question 7

Why so Spore Stain?

Answer 7

Members of the anaerobic genera Clostridium and Desulfotomaculum and the aerobic genus bacillis are spores

used to look at spores

Question 8

Spore Procedure

Answer 8

1. 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
2. Remove slides from hot plate, cool, and wash under running tap water
3. Counterstain with safranin for 30 seconds
4. Wash with tap water
5. Blot dry with biulous paper and examine under oil immersion

Question 9

Selective Media

Answer 9

• 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

Question 10

Phenylethyl Alchohol Agar

Answer 10

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

Question 11

Crystal Violet Agar

Answer 11

selective for most gram negative microogranisms. Crystal violet dye exerts an inhibitory effect on most gram positive organisms

Question 12

7.5% sodium chloride agar

Answer 12

inhibitory to most organisms other than halophilic microorganisms.
-most useful in the detection of members of the genus Staphylococcus

Question 13

Differential/Selective Media

Answer 13

• 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

Question 14

Sometimes differential and selective media

Answer 14

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

Question 15

What are the differential and selective medias?

Answer 15

• mannitol salt agar
• MacConkey’s Agar (Coliform bacilli) and (Dysentery, typhoid, and paratyphoid bacilli
• Eosin-methylene blue agar (levine)

Question 16

Mannitol Salt Agar

Answer 16

• 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

Question 17

MacConkey agar

Answer 17

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)

Question 18

Colliform Bacilli

Answer 18

• produce acid as a result of lactose fermentation

- the bacteria exhibit a red coloration on their surface

Question 19

Dysentery, typhoid, and paratyphoid bacilli

Answer 19

are not lactose fermenters and therefore do not produce acid

-colonies appear tan and frequently transparent when grown in MacConkey agar

Question 20

Eosin-methylene blue agar

Answer 20

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

Question 21

Enriched Media

Answer 21

media that have been suplemented with highly nutritious materials like blood, serum, yeast extract for the purpose of cultivating fastidious organisms

Question 22

Blood Agar

Answer 22

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

Question 23

Gamma Hemolysis

Answer 23

-no lysis of red blood cells in result no significant change in the appearance of the medium surrounding the colonies

Question 24

Alpha Hemolysis

Answer 24

Incomplete lysis of red blood cells, with reduction of hemoglobin to methemoglobin results in a greenish halo around the bacterial growth

Question 25

beta Hemolysis

Answer 25

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

Question 26

Extracellular Enzymatic Activities of Microorangisms

Answer 26

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

Question 27

Starch Hydrolysis

Answer 27

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

Question 28

Starch Hydrolysis in this experiment

Answer 28

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

Question 29

Results in starch Hydrolysis experiment

Answer 29

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)

Question 30

Lipid Hydrolysis

Answer 30

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

Question 31

Lipid Hydrolysis Experiment

Answer 31

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