STAINING

Question 1

• Developed by Hans Christian Gram
• common technique used to differentiate two large groups of bacteria based on their different cell wall constituents.
• Facilitate visualization of the specimen by enhancing the contrast in the microscopic image with the use of a dye or stain
• Gives color; easier to see

Answer 1

Staining

Question 2

Basic dyes

Answer 2

Safranin
Methylene blue
Crystal violet

Question 2

• Make use of a single dye
• Water based or aqueous or alcohol based
• Quick and easy way to visualize cell shape, size and arrangement of bacteria
• Adheres to cell surface enabling visualization of bacteria cell

Answer 2

Simple Stains

Question 3

• colors cell nuclei red

Answer 3

Safranin

Question 3

Methods in Acid-fast Stain

Answer 3

1. Zeihl-Neelsen stain
2. Kinyoun Stain

Question 3

• turns the cell nuclei blue

Answer 3

Methylene blue

Question 4

• Used to differentiate one group of bacteria from the another

Answer 4

Differential Stains

Question 4

• Distinguishes gram positive bacteria from gram negative bacteria
• Gram positive bacteria: stain blue or purple
• Gram negative bacteria: stain red
• All cocci are gram positive bacteria except neisseria, veilonella and branhamella
• All bacilli are gram negative except corynebacterium, clostridium, bacillus and mycobacterium

Answer 4

Gram Stain

Question 4

Types of Differential Stain

Answer 4

• Gram Stain
• Acid-fast Stain

Question 4

• stains the cell nuclei purple

Answer 4

Crystal violet

Question 5

• stain used for bacteria with high lipid content in their cell wall (high mycolic acid) , hence cannot stain using Gram Stain

Answer 5

Acid-fast Stain

Question 6

o Hot method
o Requires steam bathing the prepared smear after addition of the primary dye
o Acid fast organisms will appear red on a blue background

Answer 6

Zeihl-Neelsen stain

Question 7

Special Stain

Answer 7

• Lamb (Loeffler Alkaline Methylene Blue) Stain
• Hiss Stain
• Fischer-conn stain
• Doner and Schaeffer-Fulton stain
• India Ink or nigrosine

Question 7

o Cold Method
o Oil based
o It does not utilize heat after addition of primary stain, which is oil based
o Acid fast bacilli will appear red on a green background

Answer 7

Kinyoun Stain

Question 8

• Stain bacteria, fungi and blood parasites in blood smears
• Together with carbol-fuchsin solution
• To visualize metachromatic granules

Answer 8

Lamb (Loeffler Alkaline Methylene Blue) Stain

Question 8

• A type of positive staining method that stains the capsule, slime layer and the bacterial cell with a brighter background.
• Dryer stain - stains the cell wall

Answer 8

Hiss Stain

Question 9

• Used to stain microorganisms with flagella

Answer 9

Fischer-conn stain

Question 10

• The endospore stain is a differential stain which selectively stains bacterial endospores

Answer 10

Doner and Schaeffer-Fulton stain

Question 11

• Capsule of the fungus Cryptococcus neoformans

Answer 11

India Ink or nigrosine

Question 12

• Used to grow microorganism
• Aqueous solution to which all necessary nutrients essential for growth of organisms are added
• Classified into 3 primary levels: physical, chemical composition and functional type

Answer 12

Culture media

Question 13

According to Physical State

Answer 13

1. Liquid media
2. Semi-solid media
3. Solid-media

Question 14

• broth, milk or infusions, does not solidify at temperature above the freezing point
• no gelling agents such as gelatin or agar
• suited for propagation of large number of organisms, fermentation studies and other test

Answer 14

Liquid media

Question 15

• exhibit a clot like consistency at ordinary room temperature and contain agar at a concentration of 0.5% or less that allows thickening of the media without producing a firm substance
• soft consistency like custard
• best suited for culture of microaerophilic bacteria or for study of bacterial motility

Answer 15

Semi-solid media

Question 16

• contain solidifying agent such as 1.5%-2% agar
• used for isolation of bacteria and fungi or for determining the colony characteristic of the organism under study
• 2 forms: liquifiable (or reversible) solid media; non-liquifiable (non reversible) solid media

Answer 16

Solid-media

Question 17

According to Chemical Composition

Answer 17

1. Synthetic media 2. Non-synthetic media

Question 18

* Contain chemically defined substances which are pure organic and/or inorganic compounds * Example: potato dextrose agar (pda), czapek-dox agar, oat meal agar (oma), corn meal agar (cma), beef peptone agar and nutrient agar.

Answer 18

Synthetic media

Question 19

* Contain at least one ingredient that is not chemically defined * Example: extracts of animals, plants, yeast * Can support the growth of more fastidious organisms

Answer 19

Non-synthetic media

Question 20

According to Functional Type

Answer 20

1. General purpose media 2. Enrichment media 3. Selective media 4. Differential Media 5. Transport Media 6. Anaerobic Media

Question 21

* Primary isolation of a broad spectrum of microbes and contain a mixture of nutrients that support the growth of both pathogenic and non pathogenic organisms * Exanmple: peptone water, nutrient broth nutrient agar

Answer 21

General purpose media

Question 22

* Contain complex organic substances such as blood serum and special growth factors and are designed to increase the number of microorganisms without stimulating the rest of the bacterial population

Answer 22

Enrichment media

Question 23

- contains general nutrients with 5%-10% (by volume) blood added to blood agar base

Answer 23

Blood agar

Question 24

- complete lysis of rbc complete clearing around the colonies

Answer 24

Beta hemolysis

Question 25

- incomplete lysis of rbc, greenish discoloration

Answer 25

Alpha hemolysis

Question 26

- no hemolysis; no change in medium

Answer 26

Gamma hemolysis

Question 27

o Used for fastidious microorganism such as Haemophilus spp o Heat is applied to lyse the RBC causing medium to turn brown

Answer 27

Chocolate Agar

Question 28

- difficult to grow in the laboratory because they have complex or restricted nutritional and/or environmental requirements.

Answer 28

Fastidious Bacteria

Question 29

* Contain one or more substances that encourage the growth of only a specific target microorganism and inhibit the growth of others * Design to prevent the growth of unwanted contaminating bacteria or commensals so only target bacteria will grow

Answer 29

Selective media

Question 30

- contains antibiotics, used to isolation of neisseria

Answer 30

Thayer-martin agar

Question 31

- contains 10% nacl and used for isolation of staphylococcus aureus

Answer 31

Mannitol salt agar

Question 32

* Allow the growth of several types of microorganism * Designed to show visible differences among certain groups of microorganisms * Allow the growth of more than one target microorganism that demonstrates morphologic variations in colony morphology

Answer 32

Differential Media

Question 33

- promotes growth of gram negative bacteria, primarily those belonging to the family enterobacteriaceae

Answer 33

Macconkey's agar

Question 34

- used to recover mycobacterium tuberculosis, made by incorporation of malachite green

Answer 34

Lowenstein-jensen medium

Question 34

- used for the isolation of fungi

Answer 34

Sarboraud's dextrose agar

Question 34

* For organisms that cannot survive in the presence of oxygen and require reduced oxidation-reduction potential and other nutrients like Vit K and hemin * They undergo boiling to remove dissolved oxygen * Methylene blue or resarium is added as indicator of oxidation-reduction potential * Example: Chopped meat and thioglycolate broth

Answer 34

Anaerobic Media

Question 35

* Used for clinical specimen that need to be transported to the laboratory immediately after collection * Prevent drying of specimen and inhibit the overgrowth ofcommensal and contaminating organisms * Charcoal is added to neutralize inhibitory factors

Answer 35

Transport Media