METHODS: Four hundred seventeen patients with RRMM who had previously received at least two regimens were randomly assigned (2:1) to receive aponermin, thalidomide, and dexamethasone or placebo, thalidomide, and dexamethason

 ABSTRACT

BACKGROUND: Aponermin, a circularly permuted tumor necrosis factor-related apoptosis-inducing ligand, is a potential death receptor 4/5-targeted antitumour candidate. Previous phase 1/2 studies have demonstrated the efficacy of aponermin in patients with relapsed or refractory multiple myeloma (RRMM). To confirm the superiority of aponermin plus thalidomide and dexamethasone (aponermin group) over placebo plus thalidomide and dexamethasone (placebo group) in RRMM, a randomized, double-blinded, placebo controlled phase 3 trial was performed.

METHODS: Four hundred seventeen patients with RRMM who had previously received at least two regimens were randomly assigned (2:1) to receive aponermin, thalidomide, and dexamethasone or placebo, thalidomide, and dexamethasone. The primary endpoint was progression-free survival (PFS). Key secondary endpoints included overall survival (OS) and overall response rate (ORR).

RESULTS: A total of 415 patients received at least one dose of trial treatment (276 vs. 139). The median PFS was 5.5 months in the aponermin group and 3.1 months in the placebo group (hazard ratio, 0.62; 95% confidence interval [CI], 0.49-0.78; P < 0.001). The median OS was 22.4 months for the aponermin group and 16.4 months for the placebo group (hazard ratio, 0.70; 95% CI, 0.55-0.89; P = 0.003). Significantly higher rates of ORR (30.4% vs. 13.7%, P < 0.001) and very good partial response or better (14.1% vs. 2.2%, P < 0.0001) were achieved in the aponermin group than in the placebo group. Treatment with aponermin caused hepatotoxicity in some patients, as indicated by the elevated alanine transaminase, aspartate transaminase, or lactate dehydrogenase levels (52.2% vs. 24.5%, 51.1% vs. 19.4% and 44.9% vs. 21.6%, respectively), mostly grade 1/2, transient and reversible. The main grade 3/4 adverse events included neutropenia, pneumonia and hyperglycemia. The incidence of serious adverse events was similar between the two groups (40.6% vs. 37.4%). There was no evidence that aponermin leads to hematological toxicity, nephrotoxicity, cardiotoxicity, or secondary tumors.

CONCLUSIONS: Aponermin plus thalidomide and dexamethasone significantly improved PFS, OS and ORR with manageable side effects in RRMM patients who had received at least two prior therapies. These results support the use of aponermin, thalidomide, and dexamethasone as a treatment option for RRMM patients.

TRIAL REGISTRATION: The trial was registered at http://www.chictr.org.cn as ChiCTR-IPR-15006024, 17/11/2014.

PMID:37838670 | PMC:PMC10576321 | DOI:10.1186/s12885-023-11489-8

19:04

PubMed articles on: Cardio-Oncology

Multimodal Imaging of Cancer Therapy-Related Cardiac Dysfunction in Breast Cancer-A State-of-the-Art Review

J Clin Med. 2023 Sep 29;12(19):6295. doi: 10.3390/jcm12196295.

avoided. Storage and Stability a. Store the pouches at room temperature (25 and 30oC). b. Stability of the unopened pouch is as per the expiry

 

Serology

Bacteriologic diagnosis can also be confirmed by

estimating antibodies to specific antigens of the bacteria.

Examples: VDRL and Kahn tests for syphilis. ASO for

β-hemolytic streptococci and Widal for typhoid.

Preparation of Culture Media

Anything used for preparing culture media should be

free from living organisms. All media prepared should

be sterilized according to instructions for each type of

media. pH adjustment should be correct for all media.

Since, most organisms grow at a slightly alkaline pH, it

should therefore be adjusted between pH 7.2–7.6. Time

can be saved by using dehydrated culture media: as per

the manufacturer’s instructions, weigh the dehydrated

medium, add the requisite amount of boiled distilled

water, mix the two and sterilize the solution. Given below

are methods for preparation of culture media:

Peptone Water

This medium is used for the testing of indole production,

for the preparation of sugar media, and when made highly

alkaline (pH 8.0–8.4) is used for the cultivation of Vibrio

cholerae.

Peptone 10 g

Sodium chloride 5 g

Distilled water 1000 mL

Dissolve by steaming. Adjust the pH to 7.5. Filter through

paper. Distribute in tubes or bottles. Sterilize at 15 lb

pressure for 20 minutes. The commercially available

peptone water consists of water-soluble products obtained

from lean meat or other protein materials by digestion

mainly with a proteolytic enzyme like pepsin, trypsin

or papain. The important constituents are peptones,

proteoses, amino acids and inorganic salts.

Nutrient Broth

Peptone 10 g

Sodium chloride 5 g

Meat extract 10 g

Distilled water 1000 mL.

Mix the ingredients and allow to dissolve (can be

accomplished by steaming it). Adjust pH to 7.6. Phosphates

may precipitate out and should be extricated by filtration.

Distribute the medium in large bottles and then sterilize

at 15 lb for 20 minutes. When 1% glucose is added to this

nutrient broth it becomes glucose broth.

Nutrient Agar

Agar-agar is a long chain polysaccharide substance from

certain seaweeds. It forms a firm gel in watery solution at

concentrations of about 2%. Agar alone has no nutritive

properties. It melts at about 95oC and solidifies only when

cooled.

To the nutrient broth add 2% of agar—it then becomes

nutrient agar. After addition of 2% agar, autoclave at

828 Concise Book of Medical Laboratory Technology: Methods and Interpretations

15 lb for 20 minutes. Clear with white of egg. Autoclave

and filter. Distribute into flasks and sterilize at 15 lb for

20 minutes.

Blood Agar

Melt the nutrient agar and cool to 50oC. Aseptically add

5–10% sterile defibrinated sheep (ideally) blood. Mix and

pour into petri dishes or tubes which are sloped. Bank

blood or rabbit blood may be used.

Chocolate Agar

Add blood to nutrient agar as for blood agar. Mix well and

raise the temperature to 80oC keeping well mixed. Leave

at 80oC for 10 minutes. Pour into petri dishes or tubes as

needed.

Sugar Media

Sugar media are used to study the biochemical reactions of

bacteria. To sterilized peptone water add 1% of the required

sugar and 1% Andrade’s indicator. Distribute into sterile tubes

containing inverted Durham’s fermentation tubes. Indicator

is used to study the acid formation by bacteria. If acid is

produced media becomes reddish pink. Instead of Andrades

indicator the following indicators can also be used.

Neutral red 0.25% to 1% solution—if acid is produced—

pink color.

Phenol red 0.01%—if acid is produced—yellow color.

The sugar media are sterilized by fractional sterilization

or tyndallization. The sugar may be caramelized or charred

at a temperature higher than 100oC, so it is steamed on

three consecutive days in Arnold’s steam sterilizer.

Preparation of Andrade’s Indicator

Dissolve 0.5 gram of acid fuchsin in 100 mL of distilled

water. Add 16 mL of normal sodium hydroxide (NaOH)

and leave overnight. The color should change from pink to

brownish red and then to yellow.

Hiss’s Serum Water Sugars

This is used for biochemical reaction of Neisseriae.

Corynebacterium and other organisms requiring serum

for growth.

Ox serum 1 part

Distilled water 3 part

Adjust reaction to pH 7.5 and Andrades indicator 1% and

sugar 1%. Sterilize as for peptone water/sugar media.

Loeffler’s Serum Slopes

(Used for cultivating diphtheria bacilli)

Ox serum 3 parts

Glucose broth 1% 1 part

Blood is collected from ox under sterile conditions and

serum separated aseptically. Inspissate the medium at

75oC for 1 hour.

Media for Identification of Fungi

Sabouraud’s Glucose Agar

Glucose 40 grams

Peptone 10 grams

Agar 20 grams

Water to 1000 mL

Dissolve peptone in water and adjust the pH to 5.4. Add

agar and melt it at 15 lb. for 20 minutes. Then add glucose

and sterilize by fractional sterilization.

READY TO POUR, STERILIZED POUCHED MEDIA

FOR MICROBIOLOGICAL APPLICATIONS

INSTAPREP

(Courtesy: Tulip Group of Companies)

Summary

Cultivation and isolation of bacteria from pathological

samples is many a times key to the identification of

the underlying infections. With ever-increasing strains

of resistant microorganisms, susceptibility testing to

antimicrobial agents complements selecting appropriate

drugs/drug regimens to treat infections.

Availability of microbiology testing and such procedures

being available in routine laboratories has been limited

due to the availability of dehydrated media, which can

be put to use only after substantial procedural and

preparatory requirements. INSTAPREP media are ready to

use/ready to pour and fill this long felt need using a unique

proprietary technology for routine microbiological testing.

Reagent

MICROXPRESS INSTAPREP are reagents for laboratory

use only.

INSTAPREP is a ready to pour sterilized pouched media

for microbiological applications such as cultivation/

isolation/selective growth/susceptibility tests.

Nutrient Agar

Nutrient agar is used as a general culture medium. It can

be used for maintaining microorganisms for prolonged

survival of cultures. It can be used for cultivation of nonfastidious organisms. Addition of sheep blood/or serum

makes it suitable for cultivation of related fastidious

organisms. The poured medium is light straw colored,

slightly opalescent with a pH at 7.4 ± 0.2.

Microbiology and Bacteriology 829

MacConkey Agar

MacConkey agar is the standard medium for the cultivation

of enterobacteria. It is a selective and differential medium.

It contains a bile salt to inhibit nonintestinal bacteria with

neutral red to distinguish the lactose fermenting coliforms

from the lactose non-fermenting Salmonella and Shigella

species. The poured medium is a distinct clear reddish

brown color with a pH at 7.4 ± 0.2.

Cysteine lactose electrolyte deficient (CLED) Agar

with Andrade’s Indicator

CLED agar with Andrade’s indicator is a medium of

choice, recommended for use in urinary bacteriology as it

promotes the growth of all urinary pathogens. Additionally

since, it is an electrolyte deficient medium swarming due

to Proteus species is prevented and direct colony count

is facilitated. For direct colony count, the medium is

inoculated by proper dilution of the sample. Additionally

CLED agar helps identify the organism directly from the

first isolate based on colony morphology and color within

24 hours. The poured medium is slightly opalescent

greenish/gray with a pH at 7.5 ± 0.2.

Sabouraud Dextrose Agar

Sabouraud dextrose agar is the standard agar for the

cultivation and growth of fungi; particularly those

associated with skin infections. The poured medium is light

straw colored slightly opalescent with a pH at 5.6 ± 0.2.

Mueller Hinton Agar

Mueller Hinton agar is the standard agar recommended

for susceptibility tests using antibiotic sensitivity disks.

Mueller Hinton agar is recommended by NCCLS and WHO

Committee on standardization of susceptibility testing for

determining the susceptibility of microorganisms because

of its reproducibility. The poured medium is light amber

colored to slightly opalescent with a pH of 7.3 ± 0.1.

Principle

INSTAPREP ready to pour media are presterilized

media with standard proven formulations. The pouched

media only need to be kept in boiling (100oC) water for

10 minutes and they become ready to pour into sterile

plates. A result of Tulip’s long research the INSTAPREP

pouched media accord flexibility to the laboratories,

thereby avoiding laborious preparatory steps and wastage.

INSTAPREP media also help laboratories to set up cultures

on a random basis and not to be restricted to batching of

cultures. As compared to prepoured plates and dehydrated media, variability, contamination and wastage is also

avoided.

Storage and Stability

a. Store the pouches at room temperature (25 and 30oC).

b. Stability of the unopened pouch is as per the expiry

date mentioned on the label.

Additional Material Required

Water bath (250 mL beaker) at 100oC, vertical laminar air

flow/biosafety hood with Bunsen Burner, forceps/tongs,

sterile petridishes (disposable/glass), scissors, disinfectant

(70oC alcohol), absorbent sterile gauze, plastic/glass/wire

rod for hanging pouches in water bath.

Procedures

1. Retrieve the required number of pouches from the

carton.

The organism ferments sugar and produces acid and, in certain groups, gas. Acid production is indicated by a color change of the medium, due to inclusion of a pH indicator. Gas production is shown by placing a small Durham’s tube upside down in the medium during its production. Before

 

Negative Staining

Negative Staining is a technique by which organisms

remain unstained against a dark background.

India Ink Method

A small quantity of India ink 10% nigrosin is mixed with

the material on a slide. A smear is made by means of

another slide and the preparation is allowed to dry. The

smear is examined and the spirochetes are seen as clear

transparent objects against a dark brown background.

Capsules may also be demonstrated by this method.

Motility of Bacteria

Hanging Drop Method

This method is used to observe the morphology but also

demonstrates the motility of organisms. A special slide

with a concave center is used or else a ring of plasticine

can be placed on the slide. A drop of the culture of

bacterial suspension is placed on a coverslip. Vaseline is

placed near the concave area of the slide approximately

the corners of the coverslip. The slide is placed over the

coverslip so that the drop of culture is directly under the

concave area and the Vaseline adheres to the coverslip.

The slide is then quickly inverted and placed under

the microscope. Motile organisms will be seen darting

through the medium in which they are suspended. Motility

should be differentiated from Brownian movement which

is caused by bombardment of the molecules of the fluid.

In motility, the organisms move in a definite direction,

whereas in Brownian movement they show no direction.

CULTURE

Four factors are to be taken into account

1. Media providing optimum growth

2. Temperature

3. Atmosphere

4. Cultural characteristics, e.g. size, shape and pigmentation of colonies.

Media

Media can be (a) basic (b) enrichment (c) selective, and

(d) indicator media.

1. Basic Media

These contain the necessary constituents for growth—

meat extract, peptone and salt, and these are nutrient

broth (liquid) or nutrient agar (solid). Many organisms

would grow on these types of media and need no other

factors.

2. Enrichment Media

These are used for organisms, which need an additional

source of nutrition. This can be done by adding blood

or serum to the nutrient agar or broth. An enrichment

medium used for growth of the Mycobacterium tuberculosis

contains eggs.

3. Differential and Selective Media

These media by virtue of their chemical composition

inhibit the growth of some organisms while at the same

time support the growth of others. Examples: eosin methylene blue agar and MacConkey agar contain lactose and

dye or an indicator in the decolorized state. Bacteria,

which ferment lactose with the production of acid will

produce red color or colonies with metallic sheen differentiates the lactose fermenting coliform bacilli from colonies

of lactose non-fermenting organisms. Some media, which

are used are also highly selective in their action on other

organisms. Such media as SS agar, deoxycholate citrate

agar and bismuth sulfite agar will inhibit the growth of

the majority of coliform bacilli along with many strains of

proteus and will permit the successful isolation of enteric

pathogens. Tellurite glycerin agar and mannitol salt agar

are selective media for the isolation of coagulase positive

Staphylococcus from material containing other organisms.

Phenyl-ethyl-alcohol agar is a selective medium for the

isolation of gram-positive cocci in specimens or cultures

contaminated with gram-negative organisms particularly

proteus. Infusion agar containing potassium tellurite

and blood/serum inhibits the growth of normal throat

commensals and encourages the growth of C. diphtheriae.

Some medias make use of the selective antimicrobial

activity of some antibiotics and are useful for isolating

certain pathogenic organisms from material containing mixed flora. Sabouraud dextrose agar containing

cycloheximide and chloramphenicol will support the

growth of dermatophytes and most fungi, while markedly

inhibiting the growth of many saprophytic fungi and

bacteria.

4. Indicator Media

These are largely used for biochemical reactions. The

most common example is sugar media containing various

carbohydrates such as glucose, lactose, maltose, etc.

Christensen’s urea medium is used mainly in the iden-

826 Concise Book of Medical Laboratory Technology: Methods and Interpretations

tification of Proteus, which has the ability to hydrolyze

the urea, and consequently because of the presence of

phenolphthalein in the medium, a change of color is

produced.

Temperature

Most bacteria, pathogenic in humans, give optimum

growth when incubated at body temperature, i.e.

37°C. Some saprophytes, however, grow best at lower

temperatures, even as low as 4oC (cryophilic) and others at

high temperatures. The latter are known as thermophilic

bacteria and are used in testing effectiveness of sterilization

techniques.

Atmosphere

Most organisms need oxygen for growth and are incubated

in normal atmospheric conditions. Some pathogens, e.g.

tetanus bacilli, will grow only in the absence of oxygen. This

is achieved by using McIntosh and Fildes’ jar, a thick metal or

glass jar with a metal lid which can be clamped down tightly

by bolts. On this lid are 2 holes-one an air inlet and the other

an outlet. There are also 2 electric terminals. On the underside

of the lid is a piece of asbestos saturated with palladium and

covered by wire gauze. This is connected to the terminals,

and acts as a catalyst in combining any oxygen still present

after evacuation of the jar with the hydrogen, which is passed

into the jar.

The method is given below.

1. Keep the plates upside down in the jar.

2. Place in the jar an indicator—equal parts of 10% NaOH,

6% glucose and 0.5% methylene blue, boiled until the

solution becomes colorless. It should remain colorless

throughout incubation. If it turns to its original blue

color during incubation, complete anaerobiosis

(oxygenless state) has not been achieved.

3. Tightly clamp down the lid.

4. Open the air outlet valve and close the air inlet valve.

5. Attach the apparatus to an exhaust pump, and slowly

evacuate the jar (If a glass jar is used, it should be

evacuated while enclosed in a padded box to avoid

danger of explosion).

6. Allow hydrogen obtained from hydrogen cylinders or

Kipp’s apparatus in through the inlet valve after closing

the outlet valve.

7. Attach the terminals to the main current and leave for

20 minutes. This heats the palladiumized asbestos to

assist the combination of hydrogen with any remaining

oxygen.

8. Allow a little more hydrogen in via the inlet valve.

9. Put the jar in the incubator overnight. The present day

McIntosh-Filde’s jars have room temperature catalysts

and need no electrical charge. They are left at room

temperature for 15–30 minutes before allowing more

hydrogen into the jar. There are other, less complicated

methods of achieving anaerobiosis (i.e. an oxygenless

state), e.g.

a. Boil a tube of nutrient broth and layer over it

sterile Vaseline. The boiling removes the oxygen

and the Vaseline prevents more entering as the

broth cools. The tube is inoculated using a sterile

Pasteur pipette.

b. A sterile iron nail placed in glucose broth which

has been treated as in method (1), will maintain

anaerobic conditions for some time.

c. Robertson’s cooked meat medium and Brewer’s

thioglycollate broth are frequently used in the

culture of anaerobic organisms.

Some organisms are not anaerobic, but do grow better

when the amount of oxygen has been reduced. One simple

technique is to place the plates in a tin or wide mouthed

bottle with a tight fitting lid. A candle is lit inside the

container and the lid replaced firmly. The candle flame

will use off the oxygen and give an atmosphere of 5–10%

CO2. The container is placed in the incubator.

Cultural Characteristics

Bacteria grown artificially (in vitro) on agar plates are

described as colonies. These colonies vary in size, shape,

pigment production, and hemolysis on blood agar

depending on the type of media.

Colonies are described as:

1. Shape

Circular, regular, radiating or rhizard.

2. Surface

Smooth, rough, fine, granular shiny, dull, etc.

3. Size

Usually colonies are 2–3 mm in diameter, smaller ones

may be less than 1 mm.

4. Contiguity

Colonies may be discrete or swarming.

5. Consistency

May be mucoid, tenacious dry or adherent to the medium.

6. Pigmentation

Some organisms produce pigmented colonies (Staphylococci, Pseudomonas).

Microbiology and Bacteriology 827

7. Opacity

On nutrient agar they may be transparent, translucent or

opaque.

8. Elevation

Colonies may be raised, low convex, umbilicated or dome

shaped.

9. Media Changes

Colonial growth may bring about color changes in the

media themselves, e.g. hemolysis on blood agar by

hemolytic streptococci. With Pseudomonas, the green

pigment produced may diffuse into the medium.

Biochemical Reactions

Organisms that are alike in microscopic and cultural

characteristic are often differentiated by their reactions in

various biochemical tests.

1. Sugar Fermentation

Specific carbohydrate fermentation is a property of some

organisms when grown in sugar media. Sugars most

frequently employed are glucose, sucrose, lactose, mannite,

maltose and dulcite. Usually, these are incorporated into

peptone water, but for the more delicate organisms, Hiss’s

serum water must be used. Meningococci and gonococci

will only react in solid serum-sugar media. Each sugar

medium has a colored stopper and a set ‘color scheme’

may be established for the following sugars.

Glucose (green), Lactose (red), Sucrose (blue), Mannite

(mauve), Maltose (blue and white), Dulcite (pink).

The organism ferments sugar and produces acid and, in

certain groups, gas. Acid production is indicated by a color

change of the medium, due to inclusion of a pH indicator.

Gas production is shown by placing a small Durham’s tube

upside down in the medium during its production. Before

inoculating the medium the tube should be completely

filled with the medium. If gas is produced, small bubbles

of gas will be seen in the inverted tube.

2. Other Biochemical Tests

Organisms may further be identified biochemically by

their production of indole, change in pH (as shown by

the methyl red test), by their utilization of citrate and

by another test called the Voges-Proskauer reaction.

These 4 tests are especially useful in the differentiation of

intestinal pathogens.

Loeffler’s Flagella Mordant Tannic acid 20% aqueous 100 mL Ferrous sulfate crystals 20 g Loeffler’s Flagella Stain 10% alcoholic solution of Basic fuchsin 10 mL Distilled water 40 mL Microbiology and Bacteriology 825

 

7. Rinse in water again.

8. Stain with one of the following counterstains: Safranin,

Neutral red, or 1:10 Carbol fuchsin.

9. Rinse in water and allow it to dry by standing it

vertically, or by blotting it with filter paper.

Results

Because the gram-positive organisms retain the crystal

violet after decolorization, they appear dark blue in color.

The gram-negative organisms are decolorized and take up

the counterstain and therefore, appear pink in color.

Reagents

1. Crystal violet—0.5% solution in distilled water.

2. Iodine-(Lugol’s)—10 g iodine, 20 g potassium iodide

in 1000 mL of distilled water. Dissolve the potassium

iodide in 250 mL water and then add 10 g of iodine.

When dissolved make up to 1000 mL with distilled

water (This solution is three times stronger than Gram’s

iodine and is preferable).

3. Acetone.

4. Counterstain.

a. 1 g Neutral red

2 mL 1% Acetic acid

Distilled water to make 1000 mL

b. Safranin

1.7 g safranin

50 mL alcohol

Distilled water to make 500 mL

c. Dilute carbolfuchsin

1:10 dilution of strong carbol fuchsin.

Ziehl-Neelsen Stain

This stain is another method of categorizing certain

bacteria, depending on their ability to resist decolorization

by acid and alcohol. A very strong stain is used, basic

fuchsin in a phenol solution and heat is applied in order

that the stain can penetrate the waxy covering certain

bacteria.

Method

a. Make a smear of the material and allow to dry at room

temperature.

b. Flood the whole slide with strong carbol fuchsin and

heat gently underneath the slide until steam is seen

rising from the slide (Do not overheat, avoid boiling

of the stain).

c. Rinse in water and flood the slide with 25% sulfuric

acid. Leave this until the smear is pale pink in color.

d. Rinse in water and pour on alcohol for a few minutes.

e. Counterstain with malachite green, methylene blue or

picric acid.

f. Dry by standing the slide vertically—do not blot dry as

the tubercle organisms may get attached to the paper

and later may get transferred to another slide.

Results

The tubercle bacillus resists decolorizing by acid and

alcohol (i.e. it is both acid and alcohol fast) it will remain

bright red while all other organisms and material will take

on the color of the counterstain.

Troubleshooting (AFB-Staining)

Problem: False positive results

Possible causes Solutions

1. Sputum collected without washing the Patient should wash their mouth thoroughly while procuring sputum to minimize

mouth or in an unclean container specimen contamination with food particles, mouthwash or oral drugs.

 Patient should be asked to collect sputum in a clean container free from waxes,

inorganic materials and artefacts

 Artefacts may be mistaken for acid-fast bacilli

2. Oil immersion lens is not cleaned during Oil immersion lens should be cleaned after every observation to avoid contaminaobservation of slides ting other slides

Microbiology and Bacteriology 823

3. Contaminated water with acid-fast bacteria used Use clean, non-contaminated water for washing of slides during staining

for washing of slides during staining procedure

4. Carbol fuchsin held on the slide for long Allow the stain to stand for exactly 5 minutes with the application of heat. While

time with improper heating heating, ensure that the stain is not boiled. Heat only till steam starts rising

 from the slide. Leave the slide to cool for 2 minutes before decolorizing

5. Less decolorization done for thick smear The number of times for decolorization is to be increased for thick smears.

preparation Decolorization is to be carried out till the pink color disappears and the smear

appears colorless

Problem: False negative results

Possible causes Solutions

1. Sputum collected inadequately, i.e. only Thick yellowish green mucoid sputum collected from an early morning deep

the saliva productive cough should be used as a specimen

2. Failure to select suitable sputum portion Select a suitable portion, i.e. thick yellowish green mucoid portion of the sputum

for smear preparation preparation

3. Longer time duration given for counter- Allow the counterstain B to stain for 15–20 seconds before washing.

stain, i.e. more than 30 seconds

4. Inadequate examination of the smear Smear should be examined thoroughly from one edge to the other covering

100 fields or more

Modified Ziehl-Neelsen’s Stain

Used for leprosy where the bacteria are less acid fast. The

method is as mentioned above except that 5% Sulfuric acid

is used instead of 25%.

Reagents

Carbol fuchsin:

Basic fuchsin 10 g

Alcohol—100 mL

5% aqueous phenol—1000 mL.

Decolorizing agents:

25% sulfuric acid, or

5% sulfuric acid (for M. leprae) or

Acid-alcohol 3% HCI in alcohol.

Counterstains:

Loeffler’s methylene blue or

Malachite green—0.05% aqueous solution or

Methylene blue—0.1% aqueous solution or

Picric acid-saturated aqueous solution.

Special Stains

Used to stain flagella, capsules, spores and granules.

Stains for Diphtheria Bacillus

Ponder’s Stain

Toluidine blue 0.02 g

Glacial acetic acid 1 mL

Absolute alcohol 2 mL

Distilled water to make 100 mL.

Method

Spread the stain on the film for 1 minute and wash in tap water.

Result

Dark blue granules in pale blue bacillus.

Albert’s Stain

Solution I Toluidine blue 0.15 g

Malachite green 0.2 g

Glacial acetic acid 1 mL

95% alcohol 2 mL

Distilled water 100 mL

Dissolve the dyes in alcohol and add to the water and

acetic acid. Let stand for one day and filter.

Solution II Iodine 2 g

Potassium iodide 3 g

Distilled water 300 mL

Method

Apply solution I for 3 to 5 minutes, wash in tap water, blot

and dry. Apply solution II for one minute, wash, blot and

dry.

Result

The granules stain bluish black, the cytoplasm green and

other organism light green.

Modified Neisser’s Method

Neisser’s methylene blue

Methylene blue 1 g

Ethyl alcohol (95%) 50 mL

Glacial acetic acid 50 mL

Distilled water 1000 mL.

Method

a. Stain with Neisser’s methylene blue for 3 minutes.

b. Wash off with iodine solution used in Gram’s method

and leave some solution on the slide for 1 minute.

824 Concise Book of Medical Laboratory Technology: Methods and Interpretations

c. Wash in water and counterstain with neutral red

solution used in Gram’s method for 3 minutes.

d. Wash in water and dry.

Result

The bacilli show deep blue granules, the remainder of the

organism assumes a pink color.

Staining of Capsules

Hiss’s Method

a. Saturated alcoholic solution of basic fuchsin or gentian

violet 1 part to distilled water 19 parts

b. 20% aqueous copper sulfate solution.

Method

Place a few drops of solution (a) on slide. Heat to steaming

and leave on slide 30 seconds.

Wash off with solution (b).

Result

Capsule appears as faint blue halo around dark purple cell.

India Ink Method

The capsule is seen as a clear halo around the microorganism against the black background. This method may

be used for demonstrating cryptococci.

Staining of Spores

Modified Ziehl-Neelsen Method

1. Ziehl-Neelsen carbol fuchsin

2. Sulfuric acid 0.5% or methylated spirit

3. Loeffler’s methylene blue.

Method

1. Stain with carbol fuchsin for 5–10 minutes, heating

until steam rises.

2. Wash in tap water.

3. Decolorize with 0.5% sulfuric acid or methylated spirit.

If the acid is stronger than 1%, spores of many bacilli

are decolorized.

4. Wash in tap water. Now the smear is examined and if

both bacilli and spores are red, it is decolorized again.

If the spores alone are stained, it is counterstained. Let

the counterstain to act for 2 minutes. Wash in water,

blot and dry.

Result

The spores are stained bright red and the bacilli blue.

Staining of Spirochetes

Fontana’s Method

a. Fixative Acetic acid 1 mL

 Formalin 2 mL

 Distilled water 100 mL

b. Mordant Phenol 1 g

 Tannic acid 5 g

 Distilled water 100 g

c. Ammoniated silver nitrate

 Add 10% ammonia to 0.5% solution of silver nitrate in

distilled water until the precipitate formed just dissolves.

Now add more silver nitrate solution drop by drop until

the precipitate returns and does not redissolve.

Method

1. Treat the film 3 times, 30 seconds each time, with the

fixative.

2. Wash off the fixative with absolute alcohol to act for

3 minutes.

3. Drain off the excess of alcohol and carefully burn off

the remainder until the film is dry.

4. Pour on the mordant, heating till steam rises and allow

to act for 30 seconds.

5. Wash well in distilled water and again dry the slide.

6. Treat with ammoniated silver nitrate, heating till steam

rises, for half minute, when the film becomes brown

in color.

7. Wash well in distilled water and dry.

Result

The spirochetes are stained brownish black on a brownish

yellow background.

Staining of Fungi

Lactophenol Cotton Blue

Phenol crystals 20 g

Lactic acid 20 mL

Glycerol 40 mL

Cotton blue/methylene blue 0.05 g

Distilled water 20 mL

Dissolve the phenol crystals in the liquids by gently

heating and then add the dye.

Take a portion from the fungal growth and place it on

a drop of lactophenol cotton blue on a slide. Then place a

cover slip over the drop and press gently. Blot to remove

excess stain. Seal with varnish or nail polish.

Staining of Flagella

Loeffler’s Method

Loeffler’s Flagella Mordant

Tannic acid 20% aqueous 100 mL

Ferrous sulfate crystals 20 g

Loeffler’s Flagella Stain

10% alcoholic solution of

Basic fuchsin 10 mL

Distilled water 40 mL

Microbiology and Bacteriology 825

Method

Flood the smear with the mordant for 5 minutes. Wash

with distilled water. Add heated Loeffler’s flagella stain

and allow to act for 3 minutes. Wash with distilled water

and dry (The slides should be very clean).

Result

Organisms stain red and flagella pink.