Laboratory procedures for microbiological diagnosis

Laboratory procedures for microbiological diagnosis Include the following steps:
-Naked eye examination of Spenens
-Microscopy.
-Detection of microbial antigens.
-Isolation of microbes
- Antibiotic sensitivity
-Serology.
-Moleculor biology techniques.
-Gas – liquid chromatographic techniques.
-Skin tests.
I . Naked eye examination of specimens:-
This helps to determine whether a specimen is suithable or no.
- A saliva sample instead of an expectorated sputum sample should be discarded.
-Turbid CSF, is an immediate evidence of infection
-A foul – smelling pus specimen may suggest presence of anaerabes.
-A rice water stool sample may irdicate vibrio cholera infection
-Anchory sauce sputum sample would suggest invasive amoebiasis in lungs.
-Sulpur gramules in pus whould indicate actinomycosis.
II. Microscopy:
1-Wet preparation for light microscopy in examination of CSF, urine , body fluid for evidece of pus cells, and organisms.
-Vaginal secretion trichomonas and candida .
-Skin, nail , hair (in KaoH) -evidence of fungus
-Dark ground illumination to look for spirochates, treponema pallidum in suspected 1ry or 2ry .
2-Gram staind smear: It may help of saving life.
-Important in rapid diagnosis of bacterial menigitis on exam of CSF deposit.
-Diagnosis of strepto pneumonia in sputum smear
-Performed inserious septicemia when a blood culture bottle is flaged +ve.
- Identificstion of colonies appearing on culture media
Gram – stained smear may give + ve results al though the subsquent cultues are – ve as a result of given antibiotics
In vincent angina: stained smear the only means of diagnosis
Aacid fast stain of sputum allow ropid diag nosis of open plm. T.B demonstrating acid fast bacili. In other, clinical specimens such as urine , peritoneal fluid, CSF
Immunoflourescent microscopy is important for rapid diagnasis of viral infection e.g.
RSV in infants and children
-Herpes simplex
-CMV in urine throat swab.
-Rabies in brain biopsy speeimen
Clamydia trachomatis in conjunctival scraping
Also in serological antibody test e.g
-Fluorescent treponema antibody
-fluorescent amoebic antibody.
5-Eectron micro scopy:
Mainly used for rapid diagnosis of rota virus or herpes infection, CMV in neonatol urine specimens. As the virus voides in urine in large amount.
III- Detection of microbial antigens:
Becomes increasingly important in recent years.
- Immunoelectrophoresis . e.g Pneumococcal polysaccharide Ag. may be detected in sputum, serum , urine of patients by immunoelecto phoresis, when patients have already given antibiotics where as conventional sputum blood cultues are negative
-HbsAg. Detected using Glisa or latex test
-Cryptococcal antigen in CSF inpationts with cryptoccal meuingitis.
-Rota virus Ag. Using Elisa in feces of diorrhoeic chidren ivfected with rota v.
-Chlamydia trachmatis Ag using Elisa in congumctival scrapping in patients with active trachoma.
-Detection of meningococcal, hemophiluss, preumococcal Ag. In CSF specimens by latex
-Commercially available monoclonal specific antibodies for detection of Ag. In clinical specimens or cultures including various streptococcal, staphylococcal species, Neisseria, Candida spp. Chlamydia trachomatis, and Rotariruses, CMV, herpes, Adeno viruse, RSV, influenza viruses.
IV- Isolation of micropes
Is the most reliable way in which a diagnosis can be confirmed and for obtaining antimicrabial susceptibility results.
-Isolation of bacteria or fungus from specimens such as Blood, CSF (which are normally sterile) are easy to interpret.
-Bacterial or fungal isolation from specimens collected from sites with normal flora are often difficult to interpret.
-Choice of media is important according to type of specimen and suspected organism.
-Virological or chlamydial isolation methods. Need preparation of the cell line required for support grouth of suspected infectting virus.
For chlamydia - vero, Maccoy live
For Adeno - vero , HEP
For CMV- human diploid fibrablast.
For Influanza------ chick embryo.
V-Serology
10 days or longer has to pass before arising antibody demonotrated
in chronic ifections. E.g , Brucellosis. Serology is often available
-IgM ab may indicate recent infection. (e.g Rubella)
-ElISA technique widely used nowadays for detection of ab
Immune status of the patients immumization should be taken in considarations.
VI-Molecular biology techniques:
Increasingly important for rapid diagnosis of infectios for epidemiological investigations and for monitoring antimicrobial therapy
Also, important for research on the pathogenersis of infection, the developmental new raciness and immune therapeutic agents.

VII_Gas Liquid chromatographic techniques:
Become increasingly useful for the ropid detection of anaerobic infections
Specimens of pus from abdominal, gynecological or brain abscesses may be shawn to have multiple volatile fatty acids present which indicate anaerobic infection with a few hours of collection of the specimen of pus and this may affect decisions about the chemotheropy of infection.
VIII- Skin test:
Of limited value for diagnosis of infection ex:
-Mantaux skin test
-Histoplasmin test
-Casoni test
-Schick test.
IX-Antimicrobial susceptibility testing
1-Disc diffusion tests
Limitation of disc diffusion tests:-
-Not applied to slowly –growing, Fastidious organisms or anaerobes .
-Mycobacterial and fungus susceptibility testing requires specific techniques
- The reported sensitivity tests results not applied to clinical sites infections, e.g –salmonella Typhi to aminoglycosids.
-Not related to the achieved serum levels or body fluid levels of antibiotics.
-Bacteriostatic measures only.
-Can’t be applied to certain antibiotics such as polymyxines.
2,Dilution susceptibility testes:-
(micro) minimal inhibitory and minimal bactericidal activity methods.
Methods
-Broth dilution tests
-Agar dlution method.
Application:-
-Serious infection where endpoint concentration is ended
-Disc diffusion yield inter mediate susceptibility
-Life threatening infection due to organisms with unpredictable susceptibility pattern.
-Fastidious or slowly growing organisms.
-Failure of antibiotic therapy
-Serious infections caused by organisms susceptible only to toxic agents
Limitation
-Difficult
It needs the knowledge about the achievable level in serum or body fluid
3- Automated method
4-Antimicrobial concentration gradient methods
-A serial antibiotic dilations are incorporated into the agar.
-E test