BACTE LEC - QUIZ 1 Part 2 Flashcards
what does unstained/unfixed preparation determine
determines morphology and motility (whether Brownian movement or true motility)
types of motility
rownian movement or true motility
microscopes used in unstained/unfixed preparation
BF, DF, PC
Type of preparation
natural conditions suspended in fluid
unstained/unfixed preparation
–bombardment of molecules that cause movement
-organism wiggles but retains position
Brownian movement
-most organisms with ___ exhibit Brownian movement
flagella
type of motility
- uniform direction
- move from one position to another
*True Motility
microscope for detection of Treponema pallidum and other spirochetes
*Darkfield
suspending medium (so bacteria can move freely) in wet mount
NSS
Two types of unfixed/unstained preparation
wet mount
hanging drop
better approach to examine motility
-uses a concavity slide and petroleum jelly (serves as a sealant)
hanging drop
3 procedures in fixed/stained preparation
smearing
fixation
staining
procedure in staining that requires not be too thick nor too thin; may be a clinical specimen or cultured
smear
if smear is…
- too thick:
- too thin:
- too thick: cells are overlapping
- too thin: cells are washed out
-if specimen is liquid/from broth, an ________ is used
inoculating loop
if specimen is solid, a _________ is used
needle
purpose of fixation
prevents autolysis, retards bacterial physiologic functions thus preserving morphology
type of fixation
cell’s molecules change shape, heat prevents cells from being washed out
-used for microbes
o Heat fixation
type of fixation
– no destruction of structure
-used for blood smear
o Chemical fixation
purpose of staining
increased visibility, reveals additional information about the bacteria, be able to study the structure easily since cells are not moving
In smearing, if sample is from solid culture medium, place _____________ on slide
1-2 drops of NSS or distilled water
why place1-2 drops of NSS or distilled water on slide if sample is from solid culture
for even distribution of sx on slide
possess chromophore (organic molecules which produce specific colors)
Dye (salt)
organic molecules which produce specific colors)
chromophore
Bacteria are slightly negatively charged at pH ?.
7
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
