Mycobecterium

Miss Hina Asif

Hassaan Bin Nasir

Mustaqeem

Pooja Ropeta

Acid Fast

It is the differential staining techniques which was first

developed by Ziehl and later on modified by Neelsen. So this method is also called Ziehl-Neelsen staining techniques.

History of Acid Fast Staining

• In 1882 ROBERT KOCH reported the

discovery of the tubercle bacillus and

described the appearance of the bacilli

resulting from a complex staining

procedure.

• During the same time period several

other researchers (Ehrlich, Ziehl,

Rindfleisch, and Neelsen), intending to

improve on Koch’s method, introduced

modifications to the reagents and the

procedure.

• Franz Ziehl was the first to use carbolic

acid (phenol) as the mordant. Friedrich

Neelsen kept Ziehl’s mordant, but changed

the primary stain to the basic fuchsin .

• This method became known as the Ziehl-

Neelsen method in the early to mid 1890s.

•

• Acid fast organisms like Mycobacterium

contain large amounts of lipid substances

within their cell walls called mycolic acids.

These acids resist staining by ordinary

methods such as a Gram stain. – It can also be used to stain a few other bacteria, such as

Nocardia.

LIPID RICH CELL

WALL

• The stains used are the red colored Carbol

fuchsin that stains the bacteria and a

counter stain like Methylene blue or

Malachite green.

ProcedureACID FAST STAINING

Basic RequirementsCarbolfuchsin (Red)

Acid Alcohol

Counterstain with

Methylene Blue

Acid - Fast Cells – Red

Non Acid – Fast – Blue

Acid-fast Stain Reaction Explained

• Primary stain: The hot pink appearance of Acid-fast cells is

caused by Carbol fuchsin, the primary (first) stain, which is

driven into acid-fast cells using the heat from a water bath.

– Carbol Fuchsin is a lipid soluble, phenolic compound,

which is able to penetrate the cell wall

• Secondary stain (counterstain): The methylene

blue counterstain imparts Blue color to the

colorless nonacid-fast bacteria, but doesn't

change the color of acid-fast cells.

Application of Primary Stain

• 1. Carbol fuchsin primary stain of acid-fat stain;

2. Carbol fuchsin being applied to slide that had

been prepared with acid-fast controls and an

unknown bacteria. Blotting paper has been put

on top of the slide. Then the blotting paper is

saturated with stain and heated over water bath;

3. Clothes pins are useful for handling the slide;

4. Blotting paper is discarded and slide is rinsed.

Application of Decolorizer

• 1. Acid alcohol decolorizer for acid-fast stain; 2. Drizzle decolorizer down slide

for 10 - 15 seconds, while watching to see that stain is removed from negative

control; 3. Rinse

Application of Counterstain:

• 1. Secondary stain (counterstain), crystal violet;

• 2. Crystal violet is applied to slide and left for

one minute;

• 3. Rinse; 4. Stained acid fast slide,

Observation

Mycobacterium

Kingdom: Bacteria

Phylum: Actinobacteria

Class: Actinobacteria

Order: Actinomycetales

Suborder: Corynebacterineae

Family: Mycobacteriaceae

Genus: Mycobacterium

Species: M.tuberculosis

MYCOBACTERIUM-INTRODUCTION

• Mycobacteria are aerobic, Rod shaped and nonmotile bacteria (except

for Mycobacterium marinum, which shows motile within macrophages

• Cell wall –rich in lipids

• Very slow growing (15-20 Hr)

• They do not have capsules, and most do not form endospores.

• are characteristically acid fast.

Cell wall

• The distinguishing characteristic of

all Mycobacterium species is that the cell wall is thicker

than in many other bacteria, being hydrophobic, waxy,

and rich in mycolic acids or mycolates.

• contains a polypeptide layer, a peptidoglycan layer, and

free lipids.

• There are porins in the membrane to facilitate transport.

• Beneath the cell wall, there are layers of arabinogalactan

and peptidoglycan that lie just above the plasma

membrane.

Mycobacterial cell wall:

1-outer lipids, 2-mycolic acid, 3-polysaccharides (arabinogalactan),

4-peptidoglycan, 5-plasma membrane, 6-lipoarabinomannan (LAM),

7-phosphatidylinositol mannoside, 8-cell wall skeleton`

The high concentration of lipids in the cell wall of Mycobacterium

tuberculosis have been associated with these properties of the bacterium:

• Impermeability to stains and dyes

• Resistance to many antibiotics

• Resistance to killing by acidic and alkaline compounds

• Resistance to osmotic lysis via complement deposition

• Resistance to lethal oxidations and survival inside of

macrophages

Mycobacterium tuberculosis

complex

• M. tuberculosis

• M. bovis (subsp. bovis and caprae)

• vaccine strain M. bovis BCG (Bacille Calmette-Guérin)

• M. africanum

• M. canettii

• M. microti

• M. pinnipedii

These species are, with the exception of M. bovis BCG, considered to cause

tuberculosis (TB) in humans and animals. Despite their close genetic similarity,

these organisms differ considerably with regard to epidemiology, pathogenicity

and their host spectrum.

Habitate

• Mycobacteria are widespread organisms,

typically living in water (including tap

water treated with chlorine) and food

sources. Some, however, including the

tuberculosis and the leprosy organisms,

appear to be obligate parasites and are not

found as free-living members of the genus.

Myobacterium Division

• A natural division occurs between slowly– and rapidly–

growing species.

– Mycobacteria that form colonies clearly visible to the

naked eye within seven days on subculture are termed

rapid growers,

– - while those requiring longer periods are termed slow

growers

• Two media are used to grow MTB

1- Middlebrook's medium

-which is an agar based medium and

2- Lowenstein-Jensen medium which is an

egg based medium.

“Tuberculosis is defined as an infectious disease

caused by a bacterium; that most commonly

affects the lungs.”

It can also be a crippling and deadly disease, and

is on the rise in both developed and developing

worlds. Globally, it is the leading cause of deaths

resulting from a single infectious disease.

Currently, it kills “three million people” a year

and could claim up to 30 million lives if not

controlled.

What is Tuberculosis?

Types of Tuberculosis

Mycobacterium which is carried by humans.

Mycobacterium T.B. can present it self in the

human body in different forms effecting any

where from “the intestines, bones, joints, skin,

and the genito urinary, lymphatic, and nervous

systems.”

The primary stage of the disease may

be symptom-free, or the individual may

experience a flu-like illness. This is

called the “inactive stage.”

Within the active stage of the disease,

there might be a slight fever, night

sweats, weight loss, fatigue.

The symptoms may vary depending

on what type of tuberculosis you

contract.

Symptoms of Tuberculosis

PULMONARY

TUBERCULOSIS

INTRODUCTION

Pulmonary Tuberculosis (TB) is an infectiousdisease that mainly affect the lungsparenchyma.

TB is a contagious bacterial (M. tuberculosis)infection that mainly affects the lungsparenchyma, but may spread to other organs.

TB is an ancient disease. Signs of skeletal TB(pott disease) were evident in Europe fromNeolithic times, ancient Egypt, and in the pre-Columbian New World.

Physicians in ancient Greece called this illnessas “phthisis” reflecting its wasting character.

TB has remained an enemy of human society for all age.

TB is not only a problem for the person suffering from it

or their families but a public health problem of the entire

world.

TB spread from person to person by airborne transmission.

Infected person release droplet nuclei (1-5 micro meter in

diameter) through,

Talking

Coughing

Sneezing

Laughing

Singing

If not treated properly, TB can be fatal.

Clinical presentation of

Pulmonary TB

• Chronic cough

• Weight loss

• Pyrexia of unknown origin

• Unresolved pneumonia

• Chest pain

• fatigue

SIGNIFICANT LAB TEST

Tuberculin skin test:

Injecting a small amount of protein from tuberculosis bacteria between the derived layer of the skin (usually forearm).

Sputum examination and Cultures;

Is examined under a microscope to look for tuberculosis bacteria and used to grow the bacteria in a culture.

Interferon-gamma Blood test;

A simple blood is mixed with synthetic proteins

similar to those produced by the tuberculosis

bacteria.

If people are infected with tuberculosis

bacteria, their white blood cells produce certain

substances (interferons) in response to the

synthetic proteins.

SIGNIFICANT LAB TEST

IMAGING CONSIDERATION

Chest CT Scan

Chest X-ray

Treatment

Anti TB drugs:

Duration 9 months:

Isoniazid along with pyridoxine (vit B6)

Rifampicin

Ethambutol

Pyrazinamide