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PREPARATION AND EXAMINATION OF SPUTUM IN TUBERCULOSIS LABORATORY

Laboratory examination of sputum

 Description on the appearance of sputum

Purulent: green-looking, mostly pus

Mucopurulent: green looking with pus and mucus

Mucoid: mostly mucus

Mucosalivery: mucus with small small amount of saliva

Haemoptysis: containing blood

Preparation of sputum for AFB

In sputum preparation the container that contains the sample is check for proper labelling and also the nature of the sputum if it contains blood, pus or mucus to enable the proper identification of the organism. to prepare a smear, the purulent part of the sputum is selected and a wooden stick is use  to spread the area for about 2X3 cm, and the smear is avoided to be too thick at least if possible can read a newsprint through it. the smear is air dried in a safe place to protect it from dust and insect

Materials/ reagent  

Carbol fuchsin (filtered), Acid alcohol 3%v/v i.e.  3%v/v hydrochloric acid solution in 70%v/v alcohol

Filter paper,Water, Methylene blue, glass slide drainage rack immersion oil, timer, sputum container microscope, applicator stick, pencil

Ziel-neelson technique

Ziel-neelson use to stain mycobacterium species. Mycobacteria, unlike most other bacterial do not stain well by gram technique. They can how ever be stained with carbol fuchsin combined with phenol. The stain binds to the mycolic acid in mycobacteria cell wall after fix-heating After staining the acid decolorizing solution is applied, this removes the red dye from the background.  Cells, tissues fibres, and any organisms in the smear except mycobacterial which retain the dye and are there for referred to as acid fast bacilli. Following decolourization, the smear is counter stained with methylene blue, which stains the background material providing a contrast colour against which the AFB can be seen.

STEPS FOR THE PREPARATION

1.     after the smear have been prepared and air-dried as mention above

2.      the smear is heat-fix and dried

        Alcohol fixation is recommended when the smear has not been prepared from sodium hypochlorite(bleech) treated sputum and will not stain immediately. Mycobacterium tuberculosis is killed by bleech and during the staining process heat fixation of untreated sputum will not kill mycobacterium tuberculosis where as alcohol fixation is bactericidal.

3. The smear is covered with carbol fuchsin stain

4. heat is  applied until vapour begin to rise i.e. to about 60 degree to avoid overheating, the smear is allowed on the slide for 5minutes

5 the stain is washed off with clean water and

6 the smear is covered with 3%v/v acid alcohol for 5 minutes until the smear is sufficiently decolourized i.e. pink

7. Clean water is applied to wash off the any excess alcohol

8 the smear is then covered with methylene blue 5g/l for 1-2 minutes

9.wash off the stain

10 wipe the back of the slide and stand I to air dry in a draining slide rack.

EXAMINATION

The smear is examined microscopically using 100X oil immersion objective, when dispensing the oil, it is noted that the dispenser shouldn’t touch the smear reason been that there could be transfer of  Acid fast bacilli from one prep to another.

Result   

AFB: red straight or slightly curved, occurs singly or in small groups, may appear beaded

CELLS:  green

BACKGROUND MATERIAL: green

REPORTING OF SPUTUM SMEAR

When no AFB is seen after examining 300 filds, report the smear as ‘No AFB seen’

When very few AFB are i.e. when only 1 or two AFB are seen after examinating 100 fields, request a further specimen to examine (those AFB might have come from tap water or may be scratch of glass slide or by the use same piece of blotting paper while drying

When any red bacilli are seen, report the smear as AFB positive and give an indication of the number of bacteria present as follows( the greater the number the, the more infectious the patient)

a)      more than 10AFB/field at least in 20 fields report +++

b)      1-10AFB/field report as ++

c)      10-99AFB/100 report as +

d)     1-9AFB/100 fields report the exact number

PRECAUTIONS
1) smear for sputum is must notfix-heating.
2) over-heating is not advised
3) accuracy in timing is considered
4) ensure proper handling of chemicals such as ethanol, these is a flammable substance and must b handled with care

LIMITATION OF AFB MICROSCOPY

1.    Does no distinguish between viable and dead organism

2.    Limited sensitivity

3.    Limited specificity

Nematodes (Roundworms)

 (Roundworms)

The Nematodes are perhaps the secound largest group of organisms behind the arthropods. They are generally described as nonsegmented roundworms and are commonly found in the soil. They also inhabit fresh and salt water, and other more specialized habitats.

They are a diverse group, and it should be noted that most members of the group are not parasitic. With that being said, the parasitic forms have developed highly specific biological dependencies on particular hosts. Without theses hosts the parasite would not be able to survive. There are also some members that have obtained the ability to adapt to a variety of hosts. There are a number of Nematodes that are clinically significant to man.

They have a number of rather complex anatomical and physiological characteristics. Some important characteristics are the following:

1. The name roundworm originates from the fact that when sliced in a cross section fashion the worm will have a round shape to it.
2. Movement is through the use of a muscular system that lies just underneath a protective layer called the cuticle.
3. The cuticle is resistant to chemicals and each Nematode species has it's own unique structure and composition.
4. The digestive tract of Nematodes is divided into three main parts. An oral (buccal) cavity/esophagus; midgut; and hindgut/anus. The oral and hindgut are lined with epithelial cells extending from the cuticle while the midgut is lined with columnar cells lined with microvilli. The columnar cells in the midgut area allow for the absorption of ingested nutrients while the muscular esophagus functions in the delivery of food to the midgut.
5. Females are larger then the males.

The main Nematodes that are clinically significant to man are the following:

• Ascaris lumbricoides or "giant roundworm"
• Ancylostoma duodenale or "Old World Hookworm"
• Baylisascaris spp.
• Brugia malayi
• Brugia timori
• Dipetalonema perstans
• Dipetalonema streptocerca
• Enterobius vermicularis or "pinworm"
• Gnathostoma spp.
• Loa loa
• Mansonella ozzardi
• Necator americanus or "New World Hookworm"
• Onchocerca volvulus
• Strongyloides stercoralis or "Thread Worm"
• Toxocara canis
• Trichinella spiralis or "Trichina Worm"
• Trichostrongylus spp.
• Trichuris trichiura or "whipworm"
• Wuchereria bancrofti
• Dracunculus medinensis or Guinea Worm

RELEVANCE OF ANTICOAGULANT

ANTICOAGULANTS AND THEIR EFFECT  ON BLOOD

Introduction

An anticoagulant is a substance that prevents blood from clotting. Anticoagulants can be used in vivo

(1) As medication eg in thrombotic disorders such as deep vein thrombosis, pulmonary embolism, myocardial infarction and strokes.

(2) In medical equipments such as test tubes, blood transfusion bags and renal dialysis equipment.

Anticoagulants achieve their effect by suppressing the synthesis or function of various clotting factors that are normally present in the blood. Anticoagulants are  generally of two types.

 One type is heparin, which is a mixture of muco-polysaccharides that promote the activity of antithrombin III, a blood plasma protein that inactivates thrombin (an e nzyme that promotes clotting).

The other type consists of the coumarin derivatives, which interfere in the metabolism of vitamin K in the liver. Since vitamin K is an important element in the synthesis of various clotting factors, interference in the metabolism of that vitamin gives rise to clotting factors that are defective and hence inactive in the blood. Coumarin derivatives differ from heparin primarily in their longer duration  of action; of these, the most widely used is warfarin. A major side effect of anticoagulant therapy is hemorrhage. Anticoagulants are also used in drawing and storing blood.

EDTA (ethylene diamine tetraacetic acid)

 EDTA (ethylenediaminetetraacetic acid) is the most commonly used anticoagulant in evacuated tubes. It inhibits the clotting process by removing calcium from the blood. EDTA is able to form a complex with metal ions. 

 EDTA is synthesized on an industrial scale from ethylenediamine, formaldehyde,  and a source of cyanide (HCN or NaCN)

This chemical has been used to prevent clotting in blood

There is always need to pray but don't be lazy and expect God to send manners from heaven. Many believers today pray a funny prayer ( Oh God i need your blessing so that some one can give me money to buy my daily bread). As a christian be creative in thought in your mind and in your soul. Even in the bible it tells us that our Lord Jesus is creative by joining woods, he did not do sit down and look ministry, he was active doing the work of the Father who sent him and also working hard for a living. Brethren in the Lord try to be creative, use the talent and skill given to you to establish business, firm so you can pay your tithes, put your offering and give arms to the less privilege. Until you stop having the mind set that someone will give to you, discovering the creativity in you will be a story. Take for example the beggars that don't have defected hands, leg, nose, eyes, complete as a human, they go on begging on the street without the knowing they are creative. I pray you will be creative in you daily life even as you serve your God all through the way to your journey.

NIGERIA AT 55. 1st OCTOBER 2015

Happy birthday to my country, a country filed with milk and honey. my prayer for my country is peace, unity and progress. Long live Nigeria, Long live Rivers state, Long live emolga Long live rumuewhore odegu. Truly am a Nigerian. God bless my country a big shout out to nigerians happy birthday nigeria