if smear is... - too thick: - too thin:

- too thick: cells are overlapping - too thin: cells are washed out

BACTE LEC - QUIZ 1 Part 2 Flashcards by Carmina Mislang

what does unstained/unfixed preparation determine

determines morphology and motility (whether Brownian movement or true motility)

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types of motility

rownian movement or true motility

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microscopes used in unstained/unfixed preparation

BF, DF, PC

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Type of preparation

natural conditions suspended in fluid

unstained/unfixed preparation

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–bombardment of molecules that cause movement

-organism wiggles but retains position

Brownian movement

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-most organisms with ___ exhibit Brownian movement

flagella

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type of motility

uniform direction
move from one position to another

*True Motility

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microscope for detection of Treponema pallidum and other spirochetes

*Darkfield

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suspending medium (so bacteria can move freely) in wet mount

NSS

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Two types of unfixed/unstained preparation

wet mount

hanging drop

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better approach to examine motility

-uses a concavity slide and petroleum jelly (serves as a sealant)

hanging drop

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3 procedures in fixed/stained preparation

smearing
fixation
staining

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procedure in staining that requires not be too thick nor too thin; may be a clinical specimen or cultured

smear

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if smear is…

too thick:
too thin:

too thick: cells are overlapping

- too thin: cells are washed out

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-if specimen is liquid/from broth, an ________ is used

inoculating loop

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if specimen is solid, a _________ is used

needle

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purpose of fixation

prevents autolysis, retards bacterial physiologic functions thus preserving morphology

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type of fixation
cell’s molecules change shape, heat prevents cells from being washed out
-used for microbes

o Heat fixation

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type of fixation
– no destruction of structure
-used for blood smear

o Chemical fixation

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purpose of staining

increased visibility, reveals additional information about the bacteria, be able to study the structure easily since cells are not moving

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In smearing, if sample is from solid culture medium, place _____________ on slide

1-2 drops of NSS or distilled water

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why place1-2 drops of NSS or distilled water on slide if sample is from solid culture

for even distribution of sx on slide

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possess chromophore (organic molecules which produce specific colors)

Dye (salt)

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organic molecules which produce specific colors)

chromophore

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Bacteria are slightly negatively charged at pH ?.

affinity and color of nucleic acids in the nucleus (using H&E as stain)

affinity: to basic dye color: blue

affinity and color of cytoplasm (using H&E as stain))

affinity: to acidic dye color: pink

Types of Staining: – uses single dye/ one stain -end color is the color of the stain used -used for the analysis of characteristics of size, shape, and cell arrangement

Simple

Types of Staining: uses more than 1 dye -distinguishes between structure of a cell, between different types of cells by staining them with different colors -react with specific microbial structures

differential

-determines source of molecules as contaminants

gram staining

-determines the most effective antibiotic for critically ill patients

gram staining

-fundamental step in the diagnosis and treatment of diseases

gram staining

major dyes in gram staining

crystal violet and safranin

(enhances the affinity of the primary stain to the cell, promotes binding)

mordant

*Apply acetone-alcohol continuously in a ???? method. It must completely remove the stain.

running/dripping

how long must you add crystal violet

1 min

how long must you add safranin

15-30 seconds

most rapid method of bacterial detection

gram staining

most rapid method of bacterial identification

culture and sensitivity

gram + or gram - | Gram reaction - purple

gram +

gram + or gram - | Peptidoglycan layer - thick

gram +

gram + or gram - | Peptidoglycan layer - thin

gram -

gram + or gram - | teichoic acid- present

gram +

gram + or gram - | teichoic acid - absent

gram -

gram + or gram - | LPS content - present

gram -

gram + or gram - | LPS content - absent

gram +

gram + or gram - periplasmic space absent

gram +

gram + or gram - | outer membrane absent

gram +

gram + or gram - Lipid and lipoprotein content low

gram +

gram + or gram - periplasmic space present

gram -

gram + or gram - | outer membrane present

gram -

gram + or gram - | Lipid and lipoprotein content high

gram -

-primary stain in acid-fast

carbolfuchsin

decolorizer in acid-fast

acid alcohol (HCl + 95% EtOH)

mordant in acid-fast

HEAT

counterstain in acid-fast

methylene blue

also used to detect presence of Mycobacterium tuberculosis

acid-fast

-bacteria whose cell walls contain mycolic acids (render the cells resistant to decolorization)

Acid-fast

-bacteria whose cell walls do not contain mycolic acids and thus cannot resist decolorization

Non-acid-fast

cell wall has wax-like substance that is very hard to stain. But once stained, it is very hard to decolorize

Mycobacterium tuberculosis

Theories behind Gram-staining

membrane theory of knaysi isoelectric theory outer surface theory

what theory Gram-negative have high lipid content while gram-positive have high carbohydrate content and is very rich in peptidoglycan. When alcohol is added to a Gram-negative, alcohol removes the lipid from the gram-negative, thus increasing cell permeability, resulting in loss of dye-cell wall complex.

membrane theory of knaysi

what theory | Gram-negative therefore has loose network of fats and crystal violet is easily removed by the decolorizer.

membrane theory of knaysi

what theory Gram-positive cells (acidic) have low isoelectric pH (pH at which the cells have no charge) This makes cells more susceptible to entrance of dye.

isoelectric theory

contains Mg RNA

gram +

what theory outer surface of the cell wall of Gram-positive bacteria contain magnesium RNA which forms complex with CVI. This complex is insoluble with alcohol

outer surface theory