Lower Respiratory Tract Samples Biology Essay

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The oldest, simplest and commonly used form of microscopy is the light microscope. The light illuminated the specimens which are focussed using glass lenses. This can be viewed using the eye or photographic camera. Specimens are usually needed to be stained with a coloured dye to visualize its compositions. There are different stains that stain specific components of the cell such as DNA, lipids, cytoskeleton, etc. All most all microscopes today are compound microscopes that use several lenses to obtain high magnification. There has been a recent development in the use of light microscopy. This involve partly due to technical improvements which have increasingly improved the resolution more than the theoretical limit. A good example of this is fluorescence. With the help of different stains some of the common causative agent for lower respiratory tract infection can be presumptively identify.

Different stain used on lower respiratory sample

Since structures most cells of the bacteria are transparent, visualization difficult under the microscope Information about cells, tissues, and their structures within cells can be visualise in great details with the help of stain. Sometimes staining is the only way to diagnose a disease because some pathogen are slow grower or may not grow at all in routine culture. Staining techniques are easy and fast to perform and result can be obtained in a short time. That is why staining is a useful tool for the clinician in order to start antimicrobial therapy. Different types and techniques of staining can be used in lower respiratory tract infection. The most common are the following:

Gram staining

Immunofluorescence staining for Legionella spp.

Histochemical staining for Pneumocystis jiroveci

Differential staining of WBC for eosinophilia

Aid-alcohol fast bacilli (AAFB or AFB) staining

Gram Staining

In 1884, Hans Christian Gram, Danish bacteriologist, has developed the Gram staining technique (Brock, T.D., 1999). Gram staining techniques is an import method of differentiating bacterial species into two large groups. These are Gram-positive and Gram-negative. The base of the division of the group is the Gram staining reaction on the chemical and physical properties of their cell walls.

Gram staining have four processes, the primary stain which is the crystal violet, the mordant which is the iodine, the decolouriser which is made of acetone and alcohol and the counterstain which is the safranin. The Gram-positive cell walls contain higher peptidoglycan and lower lipid content. Bacteria cell walls are stained by the crystal violet. Iodine is added as a mordant to form the crystal violet-iodine complex. The fixing of the dye makes it harder to remove the dye on the cell wall. However, treatment with a decolourizer dissolves the lipid layer from the gram-negative cells. The dissolution of the lipid layer enhances the draining of the primary stain into the surrounding solvent. Allowing the counter stain to be absorbed and taking up the stain colour. In contrast, the solvent dehydrates the thicker Gram-positive cell walls, making the cell wall shrinks and closing the pores as the cell during dehydration. The diffusion of the violet-iodine complex is stop, and the bacteria remain stained and not allowing the counterstain to be absorbed.

The Gram staining techniques have simple procedure:

Prepare the slide by fixing the cells with the use of heat or exposure to methanol.

Flood the slide with Crystal violet. Let stand for 30 seconds and wash the slide with tap water.

Flood the slide with the Lugol's iodine. Let stand for 30 seconds, then wash the slide with tap water.

Decolorize the smear by applying 95% ethanol or a mixture of acetone and alcohol. Make a steady stream and make sure all the colour has come out. Then, wash with water to stop decolourisation.

Finally, counterstain with Saffranin by flooding the slide. Let stand for 30 second. Then, wash and dry. Read the smear under oil immersion.

The results for the Gram stain are the following:

Gram-positive cells will appear blue to purple.

Gram-negative cells will appear pink to red.

The advantages of Gram staining techniques are the following:

Presumptive identification and pre-culture information of the pathogen can be achieved.

Can detect different organism in the same specimen.

Evaluation of specimen and appropriate test can be done.

Rapid identification leads to aiding the clinician of their antibiotic choice for the patient.

The disadvantage of Gram staining techniques are the following:

The procedure has the tendency to have inconclusive or incorrect results due to operator technique or to the organism cellular properties.

Not all the bacteria can be stain by Gram stain like bacterial spores are difficult to stain due to the spore wall is relatively impermeable.

The procedure have less precision rate.

In relation to lower respiratory infection, the use of Gram stain is very useful especially in the most common cause of community acquired pneumonia. The characteristic of a lanceolate shape and the zone of clear around each pair, can be seen on a Gram stain of a sputum sample (.Ford, M., 2010). But on other causative agent of pneumonia, Gram stain has less importance because the morphology and characteristic have similarity with the non-pathogen organism.

Immunofluorescence staining for Legionella spp.

Immunofluorescence is a technique which the antigen in the cells are attached to a specific antibody that are chemically conjugated with fluorescent dye and can be seen under a fluorescence microscope or confocal microscope. There are two types of immunofluorescence staining methods:

Direct immunofluorescence (DFA) staining uses an antibody is labelled with fluorescence dye.

Indirect immunofluorescence (IFAT) staining uses a secondary antibody labelled with fluorochrome and used it to recognize a primary antibody.

This is a general procedure in doing a direct immunofluorescence:

Prepare cells in appropriate manner. Adjust cell suspension to a concentration of 1x106 cells/ml in Phosphate Buffered Saline pH 7.4 containing 20 mM glucose and 1% Bovine Serum Albumin.(PBS/BSA).

2 Aliquot 100 μl of cell suspension into the test-tubes.

Add appropriate volume of antibody at recommended dilution. Mix well and let it stand at room temperature for 30 minutes.

Wash with 2 ml of PBS/BSA, centrifuge at 400 g for 5 minutes. Discard supernatant.

Repeat step 4.

Resuspend cells in 0.2 ml of PBS/BSA containing 50% v/v glycerol.

Place a drop of resuspended cells on microscope slide; gently add coverslip and read using suitable FITC filter set under a fluorescence microscope.

Results interpretations of immunofluorescence staining are:

A positive indicates a significant fluorescence seen.

A negative indicates a dull green staining in all section.

The advantages of immunofluorescence staining are:

Can be used to identify different microorganisms in fresh samples or after culturing.

Fluor chromes has made it feasible to identify cells, sub-microscopic cellular composition and other distinct characteristic with a high degree of specificity.

The disadvantages of immunofluorescence staining are:

Photobleaching is destruction of a fluorophore by exposure to light. This is always problem with immunofluorescence.

It is also prone to Immunofluorescent labelling with non-specific binding.

Out of focus fluorescence is negative effect of widefield which is due to light from outside of the focal plane entering through to the detector.

Requires special equipment for visualisation and it is expensive.

This immunofluorescence staining techniques is usually use in the detection of Legionella pneumophila serogroup 1 in urine .This test is simple, quick, and very reliable but only detects Legionella pneumophila serogroup 1. Patient with the environmental source of infection cannot use this test because it cannot match and identify the specific subtypes. That is why the test has `poor predictive value' (Ford, M., 2010). In a situation where patient have a severe pneumonia; the clinician may also use urine tests for immunofluorescence staining to help prescribe the correct antibiotic.

Histochemical staining for Pneumocystis jiroveci

Histochemical stains also help to visualize, identify and classify bacteria, fungi and protozoa on their morphological characteristics. Bacteria are regularly stained with different dyes in order to visualize different properties and to intensify contrast for viewing with usual bright field microscopy. A basic principle of histochemical staining is the formation of chemical substances of a specific chemical component of cells with a stain or the development of a stain due to the reaction process. Stains or dyes are salts in which one of the ions is coloured. Basic stain has a colour which is in the positively charged ion. While in acidic stain, the colour has a negatively charged ion. Bacterial cell walls have a slight negative charge. Thus, there is an attraction between a positively charged colour ion and the negatively charged bacterial cell. A number of stains have been invented and developed to recognize capsules, spores, nuclear bodies and characteristics of the cell wall. The most common Histochemical stains for Pneumocystis jiroveci is Methenamine silver nitrate.

Methenamine silver nitrate

Methenamine silver nitrate is commonly used widely for a screen for fungal organisms. Also, it can be used to identify the yeast-like fungus Pneumocystis jiroveci which causes Pneumocystis Pneumonia (PCP) or Pneumocystosis. The mucopolysaccharide compositions of the fungal cell wall are oxidized and releasing aldehyde groups. The aldehyde groups then react with the silver nitrate. Reducing silver nitrate to metallic silver and making them visible. An outlined by the brown to black stain on the cell walls of these organisms can be observed.

Here is the procedure for Methenamine silver nitrate:

Deparaffinise and hydrate to distilled water.

2% Chromic acid, microwave Hi power for 45 seconds and make it to stand for 5 minutes.

Wash in tap water and rinse in distilled.

Put in 1% Sodium metabisulfite for 1 minute at room temperature.

Wash in tap water, rinse in distilled in 3 changes.

Working methenamine silver solution and microwave Hi power for 70 seconds. The tissue should be the colour like brown paper bag.

Agitate the slides in the hot solution and rinse in distilled water in 2 changes.

Add 0.5% Gold chloride for 1 minute or until it turns into colour grey.

Wash in distilled water.

Put 5% Hypo for 3 minutes and wash in tap water. Then, rinse in distilled.

Add working Light green for 1 minute and rinse in distilled water.

Dehydrate, clear, and put a coverslip.

The expected results are the following:

Fungi turn into black

Background will be colouring green.

The advantageous of using Methenamine silver nitrate stain are the following:

Methenamine silver nitrate stain can stains old and nonviable fungal elements more efficiently.

Speed, low-cost, and the capacity to provide a presumptive identification of the infecting fungus.

The principal and reliable means of presumptive diagnosis for patient suspected with PCP.

The disadvantageous of using Methenamine silver nitrate stain are the following:

When overstained or understained, the black colour characteristics cannot be easily demonstrated.

Sometimes you have to be sure of the morphology of the organism because there is an inclination for this stain to yield a lot of artefact from background staining.

Silver nitrate and chromic acid is toxic and avoid skin contact.

Methenamine is a hazardous agent because it is flammable solid and can also be an irritant.

Differential staining of WBC for eosinophilia

The basic principle of this differentiation is due to the distinct hemical and physical composition and characteristic of cell. Different reaction with the staining reagents producing a distinct staining characteristic. Differential staining techniques utilizes more than one stain. It can utilize separately or as combination of stain in some techniques. An example of differential stain is Giemsa satin.

Giemsa

Giemsa's stain is used in broncho-alveolar lavage sample to demonstrate the presence Pneumocystis jirovecii and `used to distinguish nuclear and/or cytoplasmic morphology of platelets, RBCs, WBCs, and parasites' (Garcia, L.S. 2001). The Giemsa stain belongs to the class of polychromatic stains which composed of a mixture of dyes of different gradation which provide fine differences in staining. The major composition being azure A and B is methylene blue prepared at an alkaline pH which spontaneously forms other dyes. This polychromatic stain was first used to stain malarial parasites. But because the property of polychromasia, it becomes very useful in staining bone marrow specimens and blood smears to distinguish between the various hemopoeitic elements.

The procedure in doing Giemsa stain:

Prepare a 1 in 10 dilution of Giemsa's stain in buffered water. Always make freshly prepared solution.

Prepare smear and let it air dry.

Fix in methanol for 60 sec

Tip off the methanol

Flood the slide with diluted Giemsa's stain and leave for 20 - 25 min

Run tap water on to the slide to float off the stain and to prevent precipitation on the smear.

Allow to air dry

Expected results:

RBCs appear pinkish grey.

Platelets appear deep pink.

WBCs have purple-blue nuclei and lighter cytoplasm.

Eosinophilic granules are bright purple-red.

Neutrophilic granules are purple.

Basophilic stippling within uninfected RBCs is blue.

Pneumocystis showing round cystic forms with halo-like borders.

The advantages: of doing Giemsa stain:

Can stain respiratory epithelial cells for cytology, as well as other common opportunistic respiratory pathogens.

Optimal as a haematology stain which can help in identifying eosinophil from other WBC's. Their presence is indicative of allergic reaction.

Differential stain designed to show virtually all nuclear and/or cytoplasmic morphology of the cell.

Can be used as tools for differential diagnosis in this patient with advanced HIV-related immunosuppression.

The disadvantages of Giemsa Stain are:

There is slight variation may emerge in the colours described above depending on the batch of stain used and how refresh it was made.

Can produce excess stain deposition on the film which can be confusing and make it difficult to differentiate cells.

Acid-alcohol fast bacilli (AAFB or AFB) staining

Ziehl-Neelsen's stain staining technique is used to demonstrate the presence of acid and alcohol fast bacilli (AAFB). Mycobacteria tuberculosis has a waxy envelopes or lipid-rich cell wall that makes them difficult to stain and decolourise. This organism is able to adhere with strong phenol dye solutions like carbol fuchsin solution. The dye remains upon succeeding differentiation in acid alcohol. Ziehl-Neelsen's staining provides morphological details and is more useful for confirming the presence of AAFB in positive cultures.

Procedure in doing Acid-alcohol fast bacilli (AAFB or AFB) staining:

Flood the slide with strong carbol fuschin.

Heat gently, and once slide is just "steaming" leave for 3 - 5 min.

Rinse well with water.

Decolourise for 2 - 3 min with a (3% v/v) acid-alcohol solution, rinse with water, then replace with fresh acid-alcohol for 3 - 4 min until the slide remains a faint pink colour.

Rinse well with water.

Counter stain with (1% w/v) methylene blue or malachite green for 30 sec.

Rinse with water and allow drying.

Read it in immersion oil under light microscope.

Expected result:

Positive: Acid fast bacilli stain red. Some may appear beaded.

Advantages of Acid-alcohol fast bacilli (AAFB or AFB) staining are the following:

Cheap, fast and simple to performed.

Less invasive.

Can increase the accuracy of this test by doing three times in three consecutive days.

High specificity and efficiency in detecting the main sources of transmission.

Disadvantages of Acid-alcohol fast bacilli (AAFB or AFB) staining are the following:

Basic fuchsin as a powder can be a potential carcinogen and inflammable. Acetic acid and hydrochloric acid is a strong irritant and a corrosive. Absolute ethanol is a flammable liquid. Methylene blue is highly toxic and an eye irritant.

False-negative errors can be attribute to poor performance of a routine staining method.

7.7b

Using a table list the different types of microorganisms that may be found in the LRT.

Indicate whether they are pathogens or normal flora and their significance based on patient group and clinical details.

Table 1.

List of organism found in the Lower Respiratory Tract and their significance.

Bacteria

Interaction with the host

Significance based on patient group and clinical details

Streptococcus pneumoniae

Pathogen

-Young adults (16-30 years)

-Older adults

-Impaired humoral immunity

-Infants and young children

- chills, fever, pleuritic chest pain,

cough and rust-colored sputum, Lobar pneumonia.

Streptococcus pyogenes (Grp A)

Pathogen

- Immunocompromised

Streptococcus agalactiae (Grp B)

Pathogen

-Infants and young children

Staphylococcus aureus

Pathogen

-Hospitalised

-Neonates

- Common in debilitated hospitalized

Patients

- Common in IV drug users

Bacillus anthracis

Pathogen

-Expose to infected animal

Nocardia sp.

Pathogen

- Immunocompromised

Enterobacteriaceae

Pathogen

-Neonatal (0-1 month)

-Hospitalised

-alcoholism

Pseudomonas aeruginosa

Pathogen

-Hospitalised

Most common hospital-acquired

pneumonia

-Vascular lesions that cause infarcts

and necrosis of the lung parenchyma

-Most common cause of lung

infections in Cystic Fibrosis

Klebsiella pneumoniae

Pathogen

Infection occurs in middle-aged,

alcoholic males usually through

aspiration

-Lobar pneumonia

-Thick currant-red jelly sputum

-Necrotizes tissue forming cavities

Acinetobacter sp.

Pathogen

-Hospitalised

Burkholderia pseudomallei

Pathogen

-Exposed to soil/water

Burkholderia mallei

Pathogen

-Exposed to soil

Yersinia pestis

Pathogen

-Close contact withinfected pets

Francisella tularensis

Pathogen

-Expose Ticks and deerflies

Hemophilus influenzae

Pathogen

-Older adults

-Impaired humoral immunity

Bordetella pertussis

Pathogen

-1-3 mo

- Strong tissue tropism for

the ciliated epithelial cells

of the respiratory tract.

-Tracheitis and bronchitis

Neisseria meningitidis

Pathogen

-Foreign travel

Legionella pneumophila

Pathogen

- Immunocompromised

-Infects older smokers, alcoholics, AIDS, cancer,

and renal transplant patients

-Lobar pneumonia

-High fever, mental confusion, proteinuria,

haematuria

-Non purulent cough

-May lead to Pontiac fever (Acute flu like

symptoms)

Bacteroides melaninogenicus

Pathogen

-Hospitalised

Fusobacterium nucleatum

Pathogen

-Hospitalised

Peptostreptococcus sp.

Pathogen

-Hospitalised

Peptococcus sp.

Pathogen

-Hospitalised

Actinomyces sp.

Pathogen

-Hospitalised

Mycobacterium tuberculosis

Pathogen

- Immunocompromised

-low socio- income

Other Mycobacterium sp.

Pathogen

- Immunocompromised

Mycoplasma pneumoniae

Pathogen

-Children (5-15 years)

-Young adults (16-30 years)

-Interstitial pneumonia

-Fever less pronounced

-Mild dry cough

-Affects young adults, prisons and military bases

-Chest X-Ray often looks worse than symptoms suggest

Chlamydia trachomatis

Pathogen

Infants (1-6 months)

Chlamydia psittaci

Pathogen

-Close contact with bird

- asymptomatic to severe bronchopneumonia with localized infiltration of inflammatory cells, necrosis, and haemorrhage

Chlamydia pneumoniae

Pathogen

-Age >5 yo

Coxiella burnetii (Q-fever)

Pathogen

-Exposed to farm environment

Viruses

Influenza

Pathogen

-Impaired cellular immunity

Parainfluenza

Pathogen

-Impaired cellular immunity

Cytomegalovirus

Pathogen

-Impaired cellular immunity

Adenovirus

Pathogen

-Impaired cellular immunity

Epstein-Barr Virus

Pathogen

-Impaired cellular immunity

Herpes Simplex Virus

Pathogen

-Impaired cellular immunity

Varicella-Zoster

Pathogen

-Impaired cellular immunity

Coxsackievirus

Pathogen

-Impaired cellular immunity

Measles

Pathogen

-Impaired cellular immunity

Rhinovirus

Pathogen

-Impaired cellular immunity

Respiratory Syncytial Virus

Pathogen

-Infants (1-6 months)

-Children (6 months-5 years)

-Impaired cellular immunity

Fungi

Aspergillus sp.

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

Mucorales sp

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

Candida sp.

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

Histoplasma capsulatum

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

Blastomyces dermatitidis

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

Cryptococcus neoformans

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

Coccidioides immitis

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

Paracoccidioides brasiliensis

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

Pneumocystis jiroveci/carinii

Pathogen

- Immunocompromised

- granuloma formation, scarring, calcification, and cavity formation

-Frothy, eosinophilic, edema fluid that

blocks oxygen exchange.

-CXR reveals a diffuse interstitial

pneumonia

Parasites-Protozoa

Cryptosporidium

- Immunocompromised

-Children

- low socio- income

Plasmodium falciparum

Pathogen

- Immunocompromised

-Expose to vector

Entamoeba histolytica

Pathogen

- Immunocompromised

-Low socio-income

Toxoplasma gondii

Pathogen

- Immunocompromised

-Expose to vector

Leishmania donovani

Pathogen

- Immunocompromised

-Expose to vector

Parasites-Nematodes

Ascaris lumbricoides

Pathogen

- Immunocompromised

-Low socio-income

Toxocara sp.

Pathogen

-- Immunocompromised

-Low socio-income

Ancyclostoma duodenale

Pathogen

- Immunocompromised

-Low socio-income

Parasites-Cestodes

Pathogen

-- Immunocompromised

-Low socio-income

Echinococcus granulosus

Pathogen

- Immunocompromised

-Low socio-income

There is no normal flora in the lower respiratory tract. Any organism can be significant and can be the causative agent for LRTI. The most common organism affecting the lower respiratory tract is that normal flora found in the upper respiratory tract.

Critically evaluate the various types of media used in the culture of LRTI's.

Include homogenisation and decontamination agents.

Before processing the sputum for culture, the sample should have pre-treatment. Semi-quantitative culture techniques for sputa are rapid and simple, and produce reliable results. An organism causing inflammation in the lungs is usually present in sputum in greater numbers than organisms that colonise the pharynx and contaminate the specimen as it is expectorated. Organisms are irregularly distributed in sputum and this can lead to inaccurate results. Liquefaction and thorough mixing of sputum allows uniform sampling. Homogenisation and dilution decreases the viscosity of the specimen without damaging any organisms present. The common digestive agents are dithiothreitol (DTT) and N-acethy-L-cystine (NAC). But our laboratory uses, normal saline (0.85%) as digestive agent. To achieve semi-quantitative culture, a 1:1000 dilution factor must be reach. Clear-cut results are obtained by culturing a suitable dilution of sputum. This is the procedure in doing homogenisation and dilution for sputum:

Add an equal volume of a normal saline (0.85%) to the sputum sample in

the Safety Cabinet.

Vortex to homogenise.

Transfer a 10 μl loopful of homogenised sputum into 5ml of sterile saline and vortex to mix.

Inoculate a 10 μl loopful of this dilution to each type of media plate.

For sample of obtained by bronchoscopy like Bronchoalveolar Lavage samples (BALs), a pre-treatment procedure is also done. But instead of doing dilution, concentration is done. This is because in this sample, the contaminating organism and any pathogen present are diluted with the saline used in bronchoscopy. Concentration is done by centrifugation and the supernatant is discarded while the deposits processed for microscopy and culture. This is how to do concentration:

Centrifuge BAL at 1200 xg for 10 mins.

Tip off all but 0.5 ml of supernatant and re-suspend centrifuged deposit in remaining fluid.

Using a sterile 10 μl loop inoculate each agar plate with the specimen.

Bacteria and fungi that can be found in lower respiratory tract infection can be isolated in routine culture media. But some needs more special requirements for growth like M. pneumonia while others does not grow at all like P. jiroveci. That is why like these two mention organism, are diagnosed by microscopy and serological test. A decontamination procedure is need in some culture media because some culture medium cannot completely inhibit all contaminants. Some common decontamination agents are NaOH, Benzalkonium chloride (Zephiran) and Oxalic acid. In a routine laboratory culture, combinations of selective and non-selective media are used. These are the following colure media:

Chocolate agar- This is use for cultivation and isolation of fastidious organisms especially Haemophilus and Neisseria species. Additional antibiotics like bacitracin can increase selectivity for H. influenza.

BLood Agar- An enriched, differential media used to isolate fastidious organisms and detect haemolytic activity and can support growth of most of the pathogenic organism found in LRT. To recover S. pneumonia in blood agar and to demonstrate a draftsman characteristic in the colonies, blood agar should be incubated in an atmosphere with enriched 5-10% carbon dioxide.

McConkey agar- This is selective and differential medium for the isolation and differentiation of the isolation of coliforms in clinical sample that contain mixed microbial flora like respiratory sample because it let a preliminary grouping of enteric and other gram-negative bacteria. Growth of Gram positive organisms are inhibited by bile salts and crystal violet. Differentiation is due to lactose which renders as supplier of fermentable carbohydrate and neutral red is a pH indicator.

Sabouraud Dextrose agar - This is for cultivation of dermatophytes. The medium provide more selective for fungi by adding chloramphenicol in the media. This allows the fungi to developed diagnostic features, such as sporing structures and pigmentation. Certain pathogenic fungi strains may grow poorly or fail to grow on this medium because of the antimicrobial agents added into a medium.

Burkholdera cepacia agar- This is selective Isolation for Burkhodera cepacia. Sputum samples from suspected patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD) are required to have inoculation for this media. Due to different nutritional need, some strains may grow poorly or fail to grow on this medium. Now and then, other bacteria maybe resistant to the selective agents will grow on these media.

Legionella CYE agar-This is used for isolation and cultivation of Legionella species. Yeast extract is the source of vitamins especially for the B-group. L-Cysteine is a nutritional source requirement. Toxic metabolites as a by-product of bacterial growth are absorbed by activated charcoal as a protective agent neutralizing. Then, hydrogen peroxide which is a toxic metabolic product decomposes and produces CO2 which modify surface tension. Growth is usually demonstrated within 3 - 4 days but it can reach up to 2 weeks.

Bordetella pertussis selective medium - A non-blood containing medium that used a charcoal medium for the isolation and cultivation of Bordetella pertussis and Haemophilus influenzae. According to Proom, Bordetellae needs nicotinic acid as important requirement for growth (Proom H., 1955). With the addition of cephalexin, it inhibits most contaminants including MRSA, Haemophilus influenzae and most coliforms. This need a prolong incubation up to 7 days.

Lowenstein-Jensen medium- This is used for isolation and differentiation of Mycobacterium spp. with fresh egg and glycerol based. Glycerol and the egg is the source of fatty acids and protein which is required for the metabolism of Mycobacteria. The solid medium is produce by the coagulation of the egg albumin during sterilization. Sodium Citrate and Malachite Green are selective agents to inhibit growth of most contaminants and encouraged early growth of Mycobacteria. Negative culture results do not rule out an active mycobacterial infection.

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