DIFFERENTIAL STAINING METHODS Flashcards
Stains
Gram stain
Acid fast stain (Ziehl-Neelsen Method)
Gram stain:
• Crystal violet
• Gram’s iodine
• Acetone alcohol or 95% Ethyl alcohol
• Safranin
Acid fast stain (Ziehl-Neelsen Method):
Carbol fuchsin
Acid alcohol
Methylene blue
refers to the form or shape of the cell as seen through the microscope.
Bacterial morphology
Bacteria come in various sizes and shapes.
Most bacteria range from_____ in diameter and from_____ in length.
0.2-2.0 um
2-8 um
Bacteria
There are three (3) forms generally recognized on the basis of the shape of the bacteria.
Cocci
Rods
Spiral
These are usually round in shape but can be oval, elongated, or flattened on one side.
Cocci
“Cocci” means, “_____” in Greek.
berry
When_____ divide to reproduce, the cells can remain attached to one another.
cocci
Cocci
Genus
Staphylococcus
Diplococcus
Streptococcus
Sarcina
Staphylococcus
irregular or grapelike-like clusters
Diplococcus
in pairs
Streptococcus
in chains
Sarcina
in cuboidal pockets of eight
Rod shape bacteria divide only across their short axis, so there are fewer groupings than of cocci.
Rods
Most____ appear in single with straight, sausage, and cylindrical or elongated shapes.
Others have tapered ends, like____.
rods
cigars
Rods
Still others are oval and look so much like cocci, called,_______.
coccobacilli
Rods shaped bacteria are called “____” which means, “little sticks”.
Bacilli
Bacillus
In chains
Clostridium
Singly
Corynebacterium
palisade or Chinese letter
Rods
Genus
Bacillus
Clostridium
Corynebacterium
Curved rods resembling a comma shape are called______.
An example is_______, the causative agent of cholera.
Vibrios
Vibrio cholerae
bacteria have one or more twists; they are never straight but resembles helicoidal or corkscrew shape.
Spiral
The spiral bacteria are divided as follows.
Spirilla
Spirochetes
actual or complete spirals, helices or may resemble the appearance of a cork-screw.
Spirilla
Its body is relatively rigid.
Example is______, the agent of rat bite fever.
Spirilla
Spirillum minor
” look like spirilla but their bodies are flexible and they wiggle while moving.
Example is ______, the causative agent of syphilis
Spirochetes
Treponema pallidu
” a bacterial suspension is dried and heat fixed so that it will adhere to the
slide.
Such smear can then be stained with colored dyes.
Fixed-stained smear
Vibrio cholerae
Curved rod
Spirillum minor, the agent of rat bite fever.
Spirilla
Example is Treponema pallidum, the causative agent of syphilis
Spirochete
This staining method uses more than one type of stain to create a contrast between two or more organisms of the same or different species that are being studied.
DIFFERENTIAL STAINING
used to compare the response of the organism with different physiological characteristics to certain staining techniques.
DIFFERENTIAL STAINING
Differential staining is also called_____ because it involves the use of at least two dyes.
multiple staining
Reagents Used in Differential Staining
- Initial stain
- Mordant
- Decolorizer
- Secondary stain
- also known as the primary stain
Initial stain
it is the first stain that is applied on the specimen.
Initial stain
- any substance which will form a bridge between the cell and the initial stain, so that the cell being studied will better retain the stain.
Mordant
Types of mordant:
Physical mordant
Chemical mordant
a. - such as heat or cold
b. - such as lodine, Ferrous sulphate,
Tannic acid
Physical mordant
Chemical mordant
- substance used to remove the initial stain.
Decolorizer
This will help in differentiating the physiological characteristic or some special structure that are present in the cell
Secondary stain
also known as the counterstain.
Secondary stain
It is the stain that is applied to the decolorized cell or cell parts.
Secondary stain
was developed in 1884 by the Danish bacteriologist Hans Christian Gram.
It is one of the most useful staining procedures because it classifies bacteria into two large groups: gram positive and gram-negative bacteria.
Gram stain
Gram stain was developed in 1884 by the Danish bacteriologist
Hans Christian Gram
Steps in Gram staining:
- A heat-fixed smear is covered with a basic purple dye, usually_____.
- After a short time, the purple dye is washed off and the smear is covered with______. When the iodine is washed off, both gram (+) and gram (-) bacteria appear______.
- The slide is washed with alcohol or an alcohol acetone solution. This solution is a______, which removes the purple from the cells of some species but not from others.
- The alcohol is rinsed off, and the slide is then stained with_____, a basic red dye. The smear is washed again, blotted dry, and examined microscopically.
crystal violet
iodine (mordant)
dark violet or purple
decolorizing agent
safranin
Bacteria that retain the dark violet or purple color are classified as______ while bacteria that lose the dark violet or purple color after decolorization are classified as______.
gram positive
gram-negative.
Different kinds of bacteria react differently to the Gram stain, because:
1. Structural differences in their cell walls affect the retention or escape of a combination of crystal violet and iodine, called the _____
2. ______ bacteria have a thicker peptidoglycan cell wall than ______
bacteria.
3. Gram (-) bacteria contain a layer of ______ as part of their cell wall.
crystal violet-lodine
complex
Gram (+)
gram (-)
lipopolysaccharide
Gram stain
When applied to both gram (+) and gram (-) cells, crystal violet and then iodine readily enter the cells.
Inside the cells, the crystal violet and iodine combine to form______.
This complex is larger than the crystal violet molecule that entered the cells, and, because of its size it cannot be washed out of the intact peptidoglycan layer of_____ cells by alcohol.
CV-I complex
gram (+)
Gram stain
• Consequently, grams (+) cells retain the color of the crystal violet dye. In gram (-) cells, however, the alcohol disrupts the_______, and the CV-I complex is washed out through the thin layer of peptidoglycan.
” As a result, gram (-) cells are colorless until counterstained with_____,
after which they are pink.
outer lipopolysaccharide layer
safranin
_____cells retain the dye and remain purple.
______cells do not retain the dye; they are colorless until counterstained with a red dye, after which they appear pink.
Gram (+)
Gram (-)
Errors in Gram Staining
Gram (+) becomes; Gram (-)
• Acidic Gram’s iodine, Insufficient iodine
•Aging, dying, autolysis, overheating.
•Removal of MgRNA with precipitation fror bile salts in the culture media
•Low concentration of crystal violet
•Over decolorization
•Excessive washing, excessive counterstaining
Errors in gram staining
Gram (+) becomes Gram (+)
• Inadequate decolorization
• Thick smears
The cell wall of some organisms, such as those belonging to the Genus Mycobacterium, contains a substantial amount of_____.
lipids
are very difficult to stain because it resists penetration of the primary dye into the cellular cytoplasm.
Lipids
Lipids are hard to penetrate
Because of this factor, application of heat or prolonging the exposure of the smear with the primary dye is needed.
Once stained, these organisms retain the stain so that even acid alcohol could not remove it. Hence, they are referred to as_____.
acid fast
Steps in Acid-Fast staining:
A.______ is applied to a fixed smear, and the slide is gently____ for several minutes.
B. Slide is cooled and washed with water.
C. Smear is next treated with_____, which removes the red stain from bacteria that are not acid-fast.
D. The smear is then stained with a_____ counterstain. Nonacid-fast cells appear_____ after application of the counterstain.
Red dye carbolfuchsin; heated
acid alcohol, a decolorizer
methylene blue
blue
The______ microorganisms retain the red color because the carbolfuchsin is more soluble in the cell wall lipids than in the acid-alcohol.
acid-fast
• In ______bacteria, whose cell wall lacks the lipid components, the carbolfuchsin is rapidly removed during decolorization, leaving the cells colorless.
non-acid fast
