LABORATORY MODULE 3. BACTERIAL STAINS

What is the purpose of staining in bacteriology?

To observe and appreciate the appearance, to differentiate one organism from another, helps identification of organisms and its special structure

What are the general rules in bacterial stains?

Basic stain should be used, young culture should also be used

This type of stain only uses one type of stain or only has one solution

Simple stain

What are the simple stains in BACTERIOLOGY

Methylene blue and Victoria Blue Dye

What is an example of stain that can be used as a simple stain and as a special stain?

Methylene Blue

Methylene Blue and Victoria Blue Dye stains the ______ of the bacteria

Cell wall

What type of stain stains the background while the bacteria itself remains colorless?

Indirect Stain

Indirect stain is also known as?

Relief stain and negative stain

Which of the indirect stain colors the background BLACK?

India ink and Nigrosin

What relief stain colors the background red?

Congo red

What negative stain colors the background purple?

Anthony

*Cryptococcus neoformans* are what type of fungi?

Dimorphic fungi

*C. neoformans* has 2 forms which are?

Yeast form and fungal form

What is the best way to demonstrate *C. neoformans* is to use what?

India ink in Barrs method

Negative stains are useful in demonstrating?

Capsulated organisms

This type of stain stains specific structure of the bacteria

Special stain

All of the following are capsular stains, EXCEPT:

a. Hiss

b. Neisser

c. Welch

d. Muir

e. Tyler

f. Gin

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LETTER ONLY

B

What color is the capsular stain HISS?

Pale brown

What color is the capsular stain TYLER?

Light violet

What color is the capsular stain WELCH?

Pale violet

What color is the capsular stain GIN?

Light blue

What color is the capsular stain WALDSWORTH?

Blue

All of the following are endospores, EXCEPT:

a. Bacillus anthracis

b. Clostridium tetani

c. Clostridium botulinum

d. Lactobacillus acidophilus

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D

What is the location of the spores of *Bacillus anthracis?*

Centrally located

What is the location of the spores of *Cryptosporidium botulinum?*

Subterminal spore

What is the location of the spores of *Clostridium tetani?*

Terminal spore

What is the appearance of *Bacillus anthracis?*

Bamboo fishing rod appearance

*Clostridium or Bacillus.* Which is catalase negative (-)

Clostridium

*Clostridium or Bacillus.* Which is catalase positive (+)

Bacillus

TRUE OR FALSE. Bacillus are known to be aerobic which means they do require oxygen

TRUE

TRUE OR FALSE. Cryptosporidium are known to be anaerobic which means they do not require oxygen

FALSE

All of the following characteristics are used to determine the bacteria with endospores present, EXCEPT

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a. Location of the spores

b. Oxygen requirement

c. pH requirements

d. Presence of enzyme catalase

e. Appearance

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C

What is the primary stain of Wirtz & Conklin

Malachite green

What is the counterstain of Schaeffer and Fulton?

Safranin

What is the primary stain of Dorner’s?

Carbon fuchsin

What is the counterstain of Dorner’s?

Nigrosin

What is the color of the spores when stained with Wirtz & Conklin

Green

What is the color of the spores when stained with Dorner’s

Red

Flagellar stains are all incorporated with?

Tannic Acid

Tannic acid ____ the flagella to be able to visualize flagella more

Swells

Enumerate all the flagellar stains

Gray, Fisher & Conn, Leifson

Metachromatic granules are also known as?

Babes-Ernst and Volutin

Babes-ernst is the inclusion body present in what bacteria?

Corynebacterium diphtheriae

Color of Albert in Metachromatic granules stain

Blue-black

Color of Neisser in Metachromatic granules stain

Dark blue

Color of Ljubinsky in Metachromatic granules stain

Dark violet

Color of ponder in Metachromatic granules stain

Dark Red

Color of LAMB in Metachromatic granules stain

Red

Meaning of “LAMB” in Metachromatic granules

Loeffler Alkaline Methylene Blue

What stain in metachromatic granules stain colors purple or violet

Burke’s modification of the Gram Stain

What media is used to enhance the presence of metachromatic granules?

Pi slant and Loeffler’s serum media

Halberstaedter prowazek is also known as?

Elementary bodies

Elementary body is a type of inclusion bodies containing a carbohydrate known as ________ present in the bacteria __________

Glycogen; Chlamydia trachomatis

Stains for Halberstaedter prowazek

Macchiavello, Castaneda, Gimenez

Which stains of Halberstaedter prowazek are red in color?

Macchiavello and Gimenez

_________ is an inclusion body present in __________ that does not have a glycogen

Levinthal Cole Lillie; Chlamydia psittaci

What inclusion body is present in *Yersinia pestis?*

Bipolar bodies

Stains of bipolar bodies

Wayson and Methylene Blue

What is the more preferred stain in bipolar bodies?

Wayson

Wayson is more effective in demonstrating the __________

Safety pin appearance of Yersinia pestis

Stain/s for nucleoid

Feulgen

This stains the DNA present in the nucleoid region

Feulgen

All of the following are are stains for spirochetes, EXCEPT:

a. Fontana Tribondeau

b. Levaditi SIlver Impregnation

c. Eosin

d. India ink

C

What is the color of spirochete when stained with India ink?

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a. Dark brown

b. black

c. Red

d. Blue

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Letter only

None of the choices

What are the bacteria stained with Castaneda, Machiavello, and Giemsa?

Rickettsia and Chlamydia

(differential staining) Gram staining was discovered by

Hans Christian Gram

A staining method of differentiating two large groups of bacteria

Differential staining

Differential staining stain what structure of a bacteria?

Peptidoglycan layer

The purpose of using acetone alcohol/95% alcohol in gram stain is to remove the ___________ present in ___________

Lipopolysaccharide; Gram negative

All are examples of COCCI GRAM NEGATIVE (-), EXCEPT:

a. Neisseria

b. Branhamella

c. Veillonella

d. Moraxella

e. All of the above

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Letter only

E

All BACILLI are GRAM NEGATIVE (-), EXCEPT?

Mycobacteria, Nocardia, Mycoplasma, Ureaplasma, Gram positive bacilli

What are GRAM POSITIVE BACILLI?

Corynebacterium, Clostridium, Bacillus, Lactobacillus, Listeria, Erysipelothrix, Actinomyces, Rothia, Kurthia

Which of the acid fast organism are theoretically gram positive wherein their peptidoglycan layer is thick and can resist gram staining

Mycobacteria

What are an example of bacilli that are known to be mollecutes and are cell wall deficit

Mycoplasma and ureaplasma

All spirochetes are reported as gram _______

Negative

TRUE OR FALSE. We do not do staining in spirochetes

TRUE

What is the most preferred microscope for spirochetes?

Dark field Microscope

Yeasts are known to be gram _____

Positive

Bacteria that is not applicable for gram staining are?

Mycoplasma and ureaplasma, spirochetes, Chlamydia and Rickettsia

What are the reason why gram positive sometimes becomes false gram negative

Old colonies used, over-decolorization, Using acidic iodione, penicillin, omits mordant in staining

What are the reason why gram negative sometimes becomes false gram positive

Under-decolorization and thick smear

The cell wall of Acid-Fast Organisms has _______ that caused them to be resistant to gram staining

Mycolic acid/hydroxy methoxy acid

What are the bacteria that are acid-fast organisms

Mycobacterium, Nocardia, Rhodococcus, Tsukamurella, Gordonia, Legionella micdadei

What are the protozoans that are acid-fast organisms

Cryptosporidium, Isospora, Sarcocystis, Cyclospora

For protozoans, the acid fast staining we use is?

Modified Kinyoun Method

2 Most common method of Acid Fast Staining

Ziehl Neelsen Method and Kinyoun Method

Which method is more useful for Direct Sputum Smear Microscopy (DSSM)

Ziehl Neelsen Method

This is a test used when we want to determine if there is a presence of M. tuberculosis in the patient

Direct Sputum Smear Microscopy

Decolorizer of Modified Kinyoun Method

1% Sulfuric Acid

Which of the acid fast stains has no mordant?

Rhodamine-Auramine Method

In Rhodamine-Auramine Method what color is produced when there is a presence of acid fast organism?

Bright yellow or orange fluorescence under green background

Decolorizing agent in Rhodamine-Auramine method

0\.5% Acid alcohol

What specimen is used in pappenheimer?

Urine

What specimen is used in baumgartens?

Tissue specimen

Pappenheimer is used to differentiate what?

M. smegmatis and M. tuberculosis

Baumgartens is used to differentiate what?

M. leprae and M. tuberculosis

Fite faraco is for the identification of?

M. leprae

Counterstain used in fite faraco?

Hematoxylin

_______ stains fungal cell wall as this has great affinity to ______

Calcofluor white; chitin

Giemsa is also used to demonstrate bacteria such as?

Borrelia