Introduction on sputum:
Trachaebronchial secretions are often collectively referred to as sputum. Sputum is constituted by plasma, water, electrolytes and mucin. Sputum is viscoelastic, i.e., some of the properties of liquid. Chemical composition revels that sputum is 90% water and only 5% solid. The solid content increases with inflammation. It also slows exfoliation of living cells.
Sputum Collection:
· Before collecting or expectorating sputum the mouth should be prerinsed and this removes contaminants from oral cavity especially.
· For most examinations, a first morning specimen is preferred as it represents the pulmonary secretions accumulated overnight.
· The specimen should be collected in a sterile disposable, impermeable container with screw cap.
· In patients who are uncooperative or cannot produce adequate sputum, induction should be tried.
· Commonly inductants are 10% sodium chloride, acetylcyteine and sterile or distilled water aerosols.
Sputum Culture:
For routine cultures, a loopful of the sputum is inoculated onto one or two blood agar, chocolate agar, MacConkey's agar and thioglycolate broth.
Saturday, October 25, 2008
Wednesday, October 22, 2008
Specimen Collection
A disease can be diagnosed partly by their signs and symptoms. But this does not reveal the full information about the causative organisms and the disease severity. So, to identify the disease causing organism, specimens are collected accordingly. This includes many rules and measures before collection. A specimen must be always collected under aseptic conditions. The different types of specimens and their respective containers are discussed below;
A Quality specimen/sample:
A sample is considered as a good quality sample, when it contains only the infective organisms of the infection and not the environmental contaminations.
A sample collected at a right time of diagnosis of the disease.
A specifically collected sample.
A sample collected before any antibiotic treatment.
A sample collected in a sterile container under aseptic conditions.
Collection of Blood:
The blood consists of a fluid of complicated and variable composition, the plasma, in which suspended erythrocytes, leukocytes and platelets. By using an anticoagulant the formed elements can be separated from plasma. When blood coagulates, the fluid that remains after separation of the clot is serum.
Ways of obtaining blood:
(a) Capillary or peripheral blood:
1. blood can be taken by pricking;
è the lobe of the ear
è the palmer surfaces of the tip of the finger
è in infants, from the plantar surfaces of the heel or the great toe
2. Puncture should be about 3mm deep.
3. An edematous or a congested part should not be used.
4. If the area to be punctured is cold or cyanotic, warm it by massaging or else erroneous may obtain.
5. Clean the site with spirit or alcohols, let dry and puncture.
6. Wipe off the first drop of blood, never press out blood.
7. Having obtained the requisite amount of blood let the patient apply slight pressure over the area with sterile swab.
(b) Venous Blood:
1. Reassure the patient about what is to be done.
2. Inspect the vein, using a tourniquet.
3. Use a syringe of a size according to the amount of blood.
4. Needles of gauze 22 should be used and be 1-1½ inches long.
5. Ask the patient to open and close the first several times.
6. Under aseptic precautions puncture the vein.
7. If it is difficult, first the skin around the vein is punctured and then the vein.
8. Make sure that the bleeding has stopped before the patient leaves.
9. Transfer blood from syringe into the container gently (not through the needle).
Monday, October 6, 2008
Determination of Bacteriocin Activity
To determine the bacteriocin antagonistic activity two methods has been performed. The first method was the plate activity assay and the second was well diffusion method.
(i) Plate Activity Assay:
MRS medium was prepared and plated under aseptic condition. The test organism (Salmonella typhi, E.coli, S.flexinariae) was swabbed over the respective MRS plate eventually. The swabbed plates were kept for 10mins to set. Using sterile inoculation loop, a loop full of direct Leuconostoc mesenteroides culture was touched over the swabbed plate and a small smear was made. The plates were incubated at 37oC for 24hrs.
(ii) Well Diffusion Method:
Mueller Hinton agar medium with 1.5% agar was prepared and plated under aseptic conditions. Using 6mm diameter well cutter, wells were made with equal distance, after the medium was set. A drop of the soft agar was dropped into the well to seal the bottom. The test organism Salmonella typhi, Escherichia coli, and Shigella flexinerrae were swabbed on the respective plates. After allowing for 10mins setting 100μl of the extracted bacteriocin was added into the well. The plates were incubated without inverting, at 37oC for 24hrs. Bacteriocin extracted by both the methods were loaded into their respective wells and checked for its antagonistic activity.
Result
(a) Well diffusion method – zone formed by bacteriocin against Salmonella typhi
(b) Well diffusion method – zone formed by bacteriocin against E.coli
(c) Plate Activity Assay
The bacteriocin was extracted and its antagonistic activity was studied against the indicator organisms by well diffusion method. The zone formed was measured and tabulated:
Salmonella typhi, zone formed – 11mm
Escherichia coli, zone formed – 6mm
Shigella flexinerrae – none
Discussion
Bacteriocin was extracted by cell free supernatant method and the crude supernatant was determined for its antagonistic activity. A well diffusion method was performed. In the activity it was observed that the bacteriocin produced by L.mesenteroides was effective against Salmonella typhi and Escherichia coli. But there was no effect against Shigella flexinerrae. The reviews say that the bacteriocins produced by L.mesenteroides are active against Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Bacillus cereus, L.monocytogenes has been considered as the major food borne pathogen and most activities were against them in food industries. Now these studies reveal the scope for bacteriocins, not only as preservatives but also as an antibiotic for many diseases and infections.
(i) Plate Activity Assay:
MRS medium was prepared and plated under aseptic condition. The test organism (Salmonella typhi, E.coli, S.flexinariae) was swabbed over the respective MRS plate eventually. The swabbed plates were kept for 10mins to set. Using sterile inoculation loop, a loop full of direct Leuconostoc mesenteroides culture was touched over the swabbed plate and a small smear was made. The plates were incubated at 37oC for 24hrs.
(ii) Well Diffusion Method:
Mueller Hinton agar medium with 1.5% agar was prepared and plated under aseptic conditions. Using 6mm diameter well cutter, wells were made with equal distance, after the medium was set. A drop of the soft agar was dropped into the well to seal the bottom. The test organism Salmonella typhi, Escherichia coli, and Shigella flexinerrae were swabbed on the respective plates. After allowing for 10mins setting 100μl of the extracted bacteriocin was added into the well. The plates were incubated without inverting, at 37oC for 24hrs. Bacteriocin extracted by both the methods were loaded into their respective wells and checked for its antagonistic activity.
Result
(a) Well diffusion method – zone formed by bacteriocin against Salmonella typhi
(b) Well diffusion method – zone formed by bacteriocin against E.coli
(c) Plate Activity Assay
The bacteriocin was extracted and its antagonistic activity was studied against the indicator organisms by well diffusion method. The zone formed was measured and tabulated:
Salmonella typhi, zone formed – 11mm
Escherichia coli, zone formed – 6mm
Shigella flexinerrae – none
Discussion
Bacteriocin was extracted by cell free supernatant method and the crude supernatant was determined for its antagonistic activity. A well diffusion method was performed. In the activity it was observed that the bacteriocin produced by L.mesenteroides was effective against Salmonella typhi and Escherichia coli. But there was no effect against Shigella flexinerrae. The reviews say that the bacteriocins produced by L.mesenteroides are active against Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Bacillus cereus, L.monocytogenes has been considered as the major food borne pathogen and most activities were against them in food industries. Now these studies reveal the scope for bacteriocins, not only as preservatives but also as an antibiotic for many diseases and infections.
Extraction of Bacteriocin
Bacteriocin extraction from Leuconostoc was done by two methods.
- Bacterial cell lysis method
- Cell free extraction method.
The activity of the supernatant recovered from bacterial cell lysis method was not detectable. It is possible that much greater force is required to disrupt L. mesenteroides, although lactobacillus casei can be disrupted by this method, as shown by Arora et al, Whereas, cell free extraction method showed a great bacteriocin activity against salmonella typhi, and Escherichia coli.
The antibacterial activity appeared to be pronounced between early logarithmic and early stationary phase. Supplementation and/or replacement of nutrients demonstrated that larger quantities of bacteriocin could be produced by addition of yeast extracts (3.0%), NaCl (1.0-2.0%), glucose (1.0%) and tween 80 (0.5%). Maximal activity in composed medium was achieved at initial pH of 5.5 and incubation period of 48hrs at 30 – 37oC.
Procedure:
(i) Bacterial cell lysis method
An overnight culture of Leuconostoc mesenteroides was centrifuged at 10,000 rpm for 20mins at 4oC. The cell was suspended in 100ml of 0.1M phosphate buffer saline (pH 7). The centrifuge and washing was repeated and the cells were pelleted once more. Then, 10ml of PBS and 5g of glass beads were added to the cell pellet and centrifuged at 200rpm and 4oC for 1hr. the suspension was centrifuged and the supernatant was recovered for the determination of antagonistic activity.
(ii) Cell Free Extraction Method
The overnight culture of Leuconostoc mesenteroides was centrifuged at 10,000rpm for 20mins, at 4oC, to obtain a cell-free solution. The supernatant solution pH was adjusted to 7.0 to exclude antimicrobial effect of organic acids. The cell free solution obtained was stirred for 2hrs at 4oC and later centrifuged at 10,000rpm for 1hr at 4oC. The precipitate was resuspended in 25ml of 0.05M potassium phosphate buffer (pH 7.0). The new precipitate was collected and used in the bacteriocin activity assay.
- Bacterial cell lysis method
- Cell free extraction method.
The activity of the supernatant recovered from bacterial cell lysis method was not detectable. It is possible that much greater force is required to disrupt L. mesenteroides, although lactobacillus casei can be disrupted by this method, as shown by Arora et al, Whereas, cell free extraction method showed a great bacteriocin activity against salmonella typhi, and Escherichia coli.
The antibacterial activity appeared to be pronounced between early logarithmic and early stationary phase. Supplementation and/or replacement of nutrients demonstrated that larger quantities of bacteriocin could be produced by addition of yeast extracts (3.0%), NaCl (1.0-2.0%), glucose (1.0%) and tween 80 (0.5%). Maximal activity in composed medium was achieved at initial pH of 5.5 and incubation period of 48hrs at 30 – 37oC.
Procedure:
(i) Bacterial cell lysis method
An overnight culture of Leuconostoc mesenteroides was centrifuged at 10,000 rpm for 20mins at 4oC. The cell was suspended in 100ml of 0.1M phosphate buffer saline (pH 7). The centrifuge and washing was repeated and the cells were pelleted once more. Then, 10ml of PBS and 5g of glass beads were added to the cell pellet and centrifuged at 200rpm and 4oC for 1hr. the suspension was centrifuged and the supernatant was recovered for the determination of antagonistic activity.
(ii) Cell Free Extraction Method
The overnight culture of Leuconostoc mesenteroides was centrifuged at 10,000rpm for 20mins, at 4oC, to obtain a cell-free solution. The supernatant solution pH was adjusted to 7.0 to exclude antimicrobial effect of organic acids. The cell free solution obtained was stirred for 2hrs at 4oC and later centrifuged at 10,000rpm for 1hr at 4oC. The precipitate was resuspended in 25ml of 0.05M potassium phosphate buffer (pH 7.0). The new precipitate was collected and used in the bacteriocin activity assay.
Thursday, October 2, 2008
Antimicrobial Protein – Bacteriocin
Bacteriocin is ribosomally synthesized antimicrobial peptides produced by most species of lactic acid bacteria. Most of them are small cationic membrane-active compounds that form pores in the target cells, disrupting membrane potentials and causing death. The production of small cationic peptides with antimicrobial activity is a defense strategy found not only in bacteria, but also in plants and animals. The antagonistic interaction between competing bacteria was described early in 1877, when Pasteur and Joubert noticed that some Escherichia coli strains interfered with the growth of Bacillus anthracis present in infected animals.
Bacteriocins was first detected in 1925 by Andre Gratia, who observed that the growth of some E.coli strains was inhibited by the presence of an antibacterial compound that he called colicin v, released into the medium by E.coli V (virulent strain). Colicin v was later characterized as a heat-stable and dialyzable peptidic compound and in 1954; Pierre Frederic found its genetic determinates in a conjugation – transmissible element similar to the F factor. The antimicrobial peptides produced by bacteria have been grouped into different classes on the basis of the producer organism, molecular size, chemical structure and mode of action.
Bacteriocin Classification:
Jack et. al considered the presence of disulphide and monosulfide (lanthionine) bonds as the basis for their classification as a landmark for their activity spectrum. Accordingly Bacteriocins were classified into four groups:
Antibiotics containing unusual post translationally modified amino acids such as dehydroalanine, dehydrobutirine, lanthionine or B-methyl lanthionine (lantibiotics);
Antibiotics containing at least one disulphide bridge essential for their activity;
Compounds with a single-SH residue that should be in a reduced form for the antibiotic to the active (thiolbiotics); and
Antibiotics without cycteine residues.
According to Klaenhammer, bacteriocins can be classified into four groups on the basis of their molecular mass, thermo stability, enzymatic sensitivity, presence of post translationally modified amino acids and mode of action.
Physical and Chemical characteristics of Bacteriocin:
To perform the bacteriocins lethal activity, it must fulfill two principal requirements:
- to be cationic
- highly hydrophobic
Most bacteriocins are active over a wide pH range (3.0 – 9.0), and while resistance to extreme pH values of 1.0 (acidocin B) and 11.0 (bavaricin A) has been observed. Most of these bacteriocins are cationic at pH 7.0. The high isoelectric point allows them to interact at physiological pH values with the anionic surfaces of bacterial membranes. Heat stability is another major feature of the bacteriocins (stable at 60oc for 120mins at pH 4.5).
They are partially inactivated by autoclaving or heating at 100oC for 120mins. Heat stability was decreased at pH 6.8. Totally inactivated by proteolytic enzymes, pronase, proteinase K, trypsin, and chemotrypsin within 25mins incubation period, whereas treatment with lipase, catalase, and α-amylase did not affect the activity of bacteriocin. The molecular mass was estimated to be 2.5 to 3.0kda based on SDS-PAGE analysis.
Bacteriocins was first detected in 1925 by Andre Gratia, who observed that the growth of some E.coli strains was inhibited by the presence of an antibacterial compound that he called colicin v, released into the medium by E.coli V (virulent strain). Colicin v was later characterized as a heat-stable and dialyzable peptidic compound and in 1954; Pierre Frederic found its genetic determinates in a conjugation – transmissible element similar to the F factor. The antimicrobial peptides produced by bacteria have been grouped into different classes on the basis of the producer organism, molecular size, chemical structure and mode of action.
Bacteriocin Classification:
Jack et. al considered the presence of disulphide and monosulfide (lanthionine) bonds as the basis for their classification as a landmark for their activity spectrum. Accordingly Bacteriocins were classified into four groups:
Antibiotics containing unusual post translationally modified amino acids such as dehydroalanine, dehydrobutirine, lanthionine or B-methyl lanthionine (lantibiotics);
Antibiotics containing at least one disulphide bridge essential for their activity;
Compounds with a single-SH residue that should be in a reduced form for the antibiotic to the active (thiolbiotics); and
Antibiotics without cycteine residues.
According to Klaenhammer, bacteriocins can be classified into four groups on the basis of their molecular mass, thermo stability, enzymatic sensitivity, presence of post translationally modified amino acids and mode of action.
Physical and Chemical characteristics of Bacteriocin:
To perform the bacteriocins lethal activity, it must fulfill two principal requirements:
- to be cationic
- highly hydrophobic
Most bacteriocins are active over a wide pH range (3.0 – 9.0), and while resistance to extreme pH values of 1.0 (acidocin B) and 11.0 (bavaricin A) has been observed. Most of these bacteriocins are cationic at pH 7.0. The high isoelectric point allows them to interact at physiological pH values with the anionic surfaces of bacterial membranes. Heat stability is another major feature of the bacteriocins (stable at 60oc for 120mins at pH 4.5).
They are partially inactivated by autoclaving or heating at 100oC for 120mins. Heat stability was decreased at pH 6.8. Totally inactivated by proteolytic enzymes, pronase, proteinase K, trypsin, and chemotrypsin within 25mins incubation period, whereas treatment with lipase, catalase, and α-amylase did not affect the activity of bacteriocin. The molecular mass was estimated to be 2.5 to 3.0kda based on SDS-PAGE analysis.
Monday, September 29, 2008
Endotoxins
Endotoxin is the lipopolysaccharide (LPS) of the outer membrane of gram negative bacteria. They are bound to bacterium and are released only when the organism lyses since it is a part of cell wall. In contrast to Exotoxin, they are
- heat stable
- toxin only at high dose
- weakly immunogenic
- generally similar, regardless of source
- cannot be toxoided
- Induces many and different pharmacological and immunological changes at low and high concentration.
- At low amounts, elicits a series of reaction : fever, activation of complement by alternative pathway, activation of macrophages and stimulation of B-cells
- In large amounts, it produces shock and hypotension and even death.
Chemistry of Endotoxin:
Bacterial LPS is composed of three parts:
è A glycophospholipid called lipid A
- is a complex array of lipid sources
- Water insoluble since it is hydrophobic
- Responsible for toxicity
- Even when paired with artificial carriers, its activity is restored.
è A Core Polysaccharide with ethanolamine and phosphate
- Common to all gram negative
- serve as carrier
è ‘O’ Antigen
- A long side chain of species specific
- Unusual polysaccharide
- serve as carriers for lipid A
Major effects of Endotoxin:
- At low concentration it sets of series of alarm reactions.
- At high range, it induces shock.
- Overlapping of these complex events depends on the amount of Endotoxin, route of infection and previous exposure of host to those substances.
- The primary target cells of Endotoxin are mono-nuclear phagocytes (monocytes, macrophages of spleen, bone marrow, lung alveoli, peritoneal cavity and kuffer cells), neutrophils, platelets and B- lymphocytes since they have specific Endotoxin receptor.
- heat stable
- toxin only at high dose
- weakly immunogenic
- generally similar, regardless of source
- cannot be toxoided
- Induces many and different pharmacological and immunological changes at low and high concentration.
- At low amounts, elicits a series of reaction : fever, activation of complement by alternative pathway, activation of macrophages and stimulation of B-cells
- In large amounts, it produces shock and hypotension and even death.
Chemistry of Endotoxin:
Bacterial LPS is composed of three parts:
è A glycophospholipid called lipid A
- is a complex array of lipid sources
- Water insoluble since it is hydrophobic
- Responsible for toxicity
- Even when paired with artificial carriers, its activity is restored.
è A Core Polysaccharide with ethanolamine and phosphate
- Common to all gram negative
- serve as carrier
è ‘O’ Antigen
- A long side chain of species specific
- Unusual polysaccharide
- serve as carriers for lipid A
Major effects of Endotoxin:
- At low concentration it sets of series of alarm reactions.
- At high range, it induces shock.
- Overlapping of these complex events depends on the amount of Endotoxin, route of infection and previous exposure of host to those substances.
- The primary target cells of Endotoxin are mono-nuclear phagocytes (monocytes, macrophages of spleen, bone marrow, lung alveoli, peritoneal cavity and kuffer cells), neutrophils, platelets and B- lymphocytes since they have specific Endotoxin receptor.
Saturday, September 27, 2008
Pharmacological toxins
Pharmacological toxins – Function by elevating or depressing normal cell functions but which do not result in death of their target cell.
Toxins that elevate cyclic AMP – Cholera toxin
These toxins raise the concentration of cyclic AMP (cAMP) without damaging the cell. The excess cAMP inhibits chemotaxis and phagocytosis, thus reducing their power to kill microorganisms. cAMP can be increased in several ways,
- some pathogens pour out cAMP themselves
- some secrete adenyl cyclase to make more cAMP from ATP
- secrete toxin that alters the activity of adenyl cyclase of host cells.
Cholera toxins (Best example)
It is an enterotoxin, a protein of mol.wt 90,000. Target tissue is the epithelium of the small intestine. It has separate A and B subunits
B component has specific affinity for the intestinal epithelial mucosa via gangliosiodic receptor.
A subunit has affinity to ADP-ribosylate of the target protein.
The target protein is part of a complex that makes cAMP. Synthesis of cAMP becomes unregulated and is made in large amount. This provokes loss of fluid and copious diarrhea which is the characteristic of Cholera.
Synthesis of cAMP
- The cyclic AMP is synthesized by the enzyme adenylate cyclase.
- It is composed of 3 proteins - Gs, R and cyclic itself.
- Gs protein is a GTP binding protein that has 2 conformational states.
- When it binds GTP, it stimulates adenylate cyclase to make cAMP.
- This effect is normally of short duration because Gs protein is also a GTPase that cleaves GTP to GDP.
- The activity of adenyl cyclase is thus determined by the balance of binding and hydrolysis of GTP by Gs protein.
- The balance of binding of GTP by Gs proteins is determined by R-protein which stimulates Gs.
- R-protein is a receptor for one of several hormones (adrenergic).
- Hence when R-protein binds one of these hormones, it interacts with Gs protein to increase its binding of GDP.
- Gs protein becomes active to stimulate adenyl cyclase.
Toxins that elevate cyclic AMP – Cholera toxin
These toxins raise the concentration of cyclic AMP (cAMP) without damaging the cell. The excess cAMP inhibits chemotaxis and phagocytosis, thus reducing their power to kill microorganisms. cAMP can be increased in several ways,
- some pathogens pour out cAMP themselves
- some secrete adenyl cyclase to make more cAMP from ATP
- secrete toxin that alters the activity of adenyl cyclase of host cells.
Cholera toxins (Best example)
It is an enterotoxin, a protein of mol.wt 90,000. Target tissue is the epithelium of the small intestine. It has separate A and B subunits
B component has specific affinity for the intestinal epithelial mucosa via gangliosiodic receptor.
A subunit has affinity to ADP-ribosylate of the target protein.
The target protein is part of a complex that makes cAMP. Synthesis of cAMP becomes unregulated and is made in large amount. This provokes loss of fluid and copious diarrhea which is the characteristic of Cholera.
Synthesis of cAMP
- The cyclic AMP is synthesized by the enzyme adenylate cyclase.
- It is composed of 3 proteins - Gs, R and cyclic itself.
- Gs protein is a GTP binding protein that has 2 conformational states.
- When it binds GTP, it stimulates adenylate cyclase to make cAMP.
- This effect is normally of short duration because Gs protein is also a GTPase that cleaves GTP to GDP.
- The activity of adenyl cyclase is thus determined by the balance of binding and hydrolysis of GTP by Gs protein.
- The balance of binding of GTP by Gs proteins is determined by R-protein which stimulates Gs.
- R-protein is a receptor for one of several hormones (adrenergic).
- Hence when R-protein binds one of these hormones, it interacts with Gs protein to increase its binding of GDP.
- Gs protein becomes active to stimulate adenyl cyclase.
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