bacterial staining

Stained preparations

— are needed to examine micro-organisms microscopically in order to study their morphology and observe their cellular constituents.

liquid, solid, clinical

Smears can be made from — or — cultures or from the — specimen.

basic stain, acidic stain

To prepare specimens to be viewed under the compound microscope there are two types of stains that are used:

basic stain

type of stain that is composed of positively charged ion that attaches to the negatively charged cell membrane

acidic stain

type of stain that is composed of negatively charged ion that is repelled by the negatively charged membrane

eosin, acid fuschin

examples of acidic stains

crystal violet, safranin

examples of basic stains

acid dyes

— is usually stain the background

simple, differential, special stains

in bacteriology, staining methods are divided into three categories:

simple stains

a staining method that impart the same color to all structures and makes use of the direct staining method

simple stains

a staining method used to demonstrate the presence of and the morphology of bacteria and cells

differential stains 

a staining method that contain more than one dye and impart different colors to various structures

gram staining, acid fast staining

examples of differential stains

special stains

a specialized staining methods to demonstrate certain bacterial components

endospore, flagella, capsule

special stains are used to highlight specific bacterial structures such as:

negative staining

Stain where the organism remains unstained against a stained background

nigrosine, india ink

Pigments commonly used in negative staining

microorganisms 

pigments of the negative staining do NOT penetrate the —, instead they obliterate the background, leaving the organisms transparent and visible in a darkened field 

bacterial cell wall 

The structure that maintains cell rigidity and integrity, determines cell shape, prevents lysis due to osmotic pressure, and may contribute to pathogenicity

peptidoglycan

Primary component of bacterial cell wall that maintains rigidity and shape

gram positive

Type of bacteria with thick peptidoglycan layer that stains purple

gram negative

Type of bacteria with thin peptidoglycan layer and outer membrane that stains pink or red

label

preparation of bacterial smear

1. — a clean glass slide with name of organism

target circle 

preparation of bacterial smear

2. make a “—” on the bottom side of the slide with a marking pen 

distilled water

preparation of bacterial smear
3. flame an inoculating loop, let it cool, and transfer 2 or 3 loopfuls of — to the center of the target circle 

bacteria 

preparation of bacterial smear

4. with an inoculation loop or needle, pick up a small amount of —. mix it well with the water and spread the mixture over a wider area of the slide 

air dry

preparation of bacterial smear

5. — the bacterial specimen on the slide

heat

preparation of bacterial smear

6. when slides are completely air-dry, — fix the bacterial specimen by passing the slide slowly over the flame twice

bacterial fixation

Process that stops or arrest the bacterial physiological processes, preserving the normal morphological state of the bacterial organelles

heating, air drying, treating with alcohol

bacterial fixation is done for the preparation of bacterial smear either through —, —, or —

bacterial fixation 

process that adhere the bacteria on the glass slide to prevent washing out during decolorization and rinsing 

crystal violet

gram staining procedure

1. cover the smear with — and let stand for 20 seconds

distilled water

gram staining procedure

2. briefly wash off the stain (crystal violet), using a water bottle of —. drain off excess water 

gram’s iodine solution

gram staining procedure

3. cover the smear with — and let it stand for 1 minute 

95% ethyl alcohol

gram staining procedure

4. pour off the gram’s iodine and flood the smear with — for 10 - 20 seconds. this step is critical. 

colorlessly 

gram staining procedure

4. decolorization has occurred when the solvent flows — from the slide 

rinsing

gram staining procedure

5. stop action of the alcohol by — the slide with water from wash bottle for few seconds

safranin

gram staining procedure

6. cover the smear with — for 20 seconds 

bibulous paper

gram staining procedure

7. wash gently for a few seconds, blot dry with — and air dry

oil immersion

gram staining procedure

8. examine the slide under —

cell wall

the gram stains works simply because there is a difference in the — between G+ and G- organisms 

mycolic acid 

mycobacterium has a waxy substance in its cell wall called — that makes it difficult to stain using a basic dye

impermeable

acid fast microorganisms are characterized by wax-like nearly — cell wall

mycolic acid, fatty acids, waxes, complex lipids

acid fast microorganisms contain — and large amounts of —, —, —

hydrophobic

acid fast is extremely —. they can’t take up stain and are highly resistant to disinfectants and dry conditions 

acid fast stain

staining that is used to distinguish mycobacterium from other types of rods

carbol fuschin

Primary stain used in acid-fast staining

phenol

carbol fuschin is a lipid-soluble and contains — which helps the stain penetrate the cell wall

basic fuschin, ethanol, phenol, distilled water

components of carbol fuschin

red

after using the primary stain in acid fast procedure, all bacteria will be color —

heat 

mordant of acid fast stain (used to force the stain on the organism)

acid alcohol

decolorizer used in acid fast stain (very strong decolorizer which strips the stain from all non-acid fast cells but does not permeate the cell wall of acid fast organisms)

methylene blue

secondary stain used in acid fast stain (to bind to any organism that did not bind to the primary stain)

endospore

a special structure made by some bacteria to store genetic material for a new cell

ordinary dyes

endospore can’t be stained with — because it don’t penetrate the wall

keratin

endospore are made of tough protein coats made of — making it highly resistant to normal staining procedures 

intramolecular disulfide

the coat of endospore is composed of a keratin-like protein containing many —bonds 

malachite green 

Primary stain used in endospore staining

heat

— is used with malachite green to force it into endospore 

water

Decolorizer used in endospore staining

safranin

secondary stain used in endospore staining that stains the cell wall of the microbe giving a contrast between the endospore and the organism

non-ionic

bacterial capsules are — so neither acidic nor basic stains will adhere to their surfaces

acidic, basic

the best way to visualize the bacterial capsule is to stain the background using an — and to stain the cell itself using a —

crystal violet

primary stain used in anthony’s capsule stain

copper sulfate

mordant used in anthony’s capsule stain 

crystal violet, white

in anthony’s capsule stain, bacterial cells and background is stained with — while the unstained capsule will appear — 

flagella stain

stain that is very tedious and delicate staining procedure 

tannic acid

Mordant used for flagella staining

visible 

flagella stain uses a mordant (tannic acid) and a stain (pararosaniline or basic fuschin) to build up the diameter of the flagella until they become —