BACTE LAB LA10 and 11 Flashcards
Visual culture characteristics of a bacterial colony on agar
plate
Colonial Morphology
what is the Smibert and Krieg Protocol
- Measure the colony diameter in millimeters
- Describe the pigmentation.
- Describe the form, elevation, and margin. Also indicate whether the colony is smooth, rough or mucoid.
- Record the opacity of the colonies
- Determine texture when tested with a needle.
Color and Pigment of Serratia marcescens
Red- Prodigiosin
Color and Pigment of Pseudomonas aeruginosa
Blue Green- Pyocyanin
Color and Pigment of Staphylococcus aureus
Yellow- Zeaxanthin
Why is colonial morphology performed
Features of the colonies may help to pinpoint the identity of the bacterium.
It gives us idea how many species are there in a specimen.
who discovered Gram Stain
Hans Christian Gram (1884)
most commonly used stain in the clinical microbiology laboratory
GRAM STAIN
Color of Gram Positive Bacteria
Blue to Purple
Color of Gram Negative Bacteria
Pink
Clinical samples are directly smeared
and stained for gram-staining
Direct Gram Staining/Direct Smear
Provides a preliminary diagnosis regarding the infectious agents and
frequently is used to direct initial therapies for the patient
Direct Gram Staining/Direct Smear
Prepared from bacterial colonies growth in culture medium
Indirect Gram Staining/Indirect Smear
Used to determine subsequent testing for identifying and characterizing the
organisms isolated from the patient specimen
Indirect Gram Staining/Indirect Smear
Principle of Gram Stain
Based on the differences in composition
of peptidoglycan of Gram (+) and Gram (-) Bacteria
cell walls, which contain
thick peptidoglycan with numerous teichoic acid cross-linkages
Gram-positive Bacteria
cell walls, which consist
of a thinner layer of peptidoglycan and
an outer lipid bilayer that is dehydrated
during decolorization
Gram-negative Bacteria
Primary Stain of the Gram Stain
Crystal Violet (hexamethyl-p-rosaniline chloride)
Crystal Violet is a basic chemical dye that consists of _____________________ charges
positive or cationic
Crystal Violet dye will adhere to the organisms whose membranes have ______________________ charges
negative or anionic
Crystal Violet Has
affinity to the _________________ layer
peptidoglycan
Mordant used for Gram Stain
Gram’s Iodine
Time of application of the Mordant
1 Minute
Time of application of the Primary Dye
1 minute
interacts with CV(+) to form large
complexes (insoluble to water) and to
replace the smaller chloride in the stain
molecule
Gram’s iodine
- Most critical part of the
procedure/component - Will differentiate the bacteria
Decolorizer
What is the Decolorizer for Gram Stain
Acetone Alcohol
Time of Application of Decolorizer
Less than 10
seconds
Effect of Acetone Alcohol to Gram (+) bacteria
Peptidoglycan is dehydrated and shrunk,
trapping the CV-I complex inside
Effect of Acetone Alcohol to Gram (-) bacteria
Damages these thin lipid walls (LPS) and allows
the stain complex to wash out
Counter Stain for Gram Stain
Safranin
Time of Application of Counter Stain
1 minute
Cells in a direct smear from a patient specimen, such as epithelial cells, white blood cells,
red blood cells, and amorphous background material, should appear ______________________ if
the Gram stain procedure was performed correctly.
pink (gram-negative)
Bordetella pertussis leave safranin on for _______________ or replace safranin with _____________________
2 MINUTES, 0.2%,
BASIC FUCHSIN
POSITIVE CONTROL FOR GRAM-STAINING
Staphylococcus aureus
NEGATIVE CONTROL FOR GRAM STAINING
Escherichia coli
gram-staining for Fungi
Hucker’s Modification (CV+ ammonium oxalate)
All cocci are gram(+) EXCEPT
NeVerMind (Neisseria,
Veillonella, Moraxella- gram negative cocci)
All bacilli are gram(-) EXCEPT
MCC-BELL
(Mycobacteria, Corynebacterium, Clostridium,
Bacillus, Erysipelothrix, Lactobacillus, Listeria)
Higher forms of organisms like ______________________ are gram (+)
Actinomyces, Nocardia,
Streptomyces, yeast and molds
All ________ organisms are reported as Gram (-)
spiral
NOT GRAM STAINED: ___________________________
Rickettsia, Chlamydia,
Mycoplasma, Ureaplasma, Spirochetes
