AT THE END OF THE COURSE, YOU WILL BE ABLE TO APPLY: MOLECULAR/CONVENTIONAL DIAGNOSTIC MICROBIOLOGY TECHNIQUE IN D’SE Dx!
NB: 1. THERE SHALL BE @ LST 3 EXAMS.
2 . NO FORMAL LECTURE NOTES WILL BE GIVEN
Qn1: Highlight the major steps involved during Gram’s stain in a diagnostic lab
Qn2: Briefly explain why Gram –ve bacteria don’t retain basic stain after decolourization. (Less than 2 lines)
Getting 2 know @other in BLS 206!
Laboratory Diagnosis of Infection Ask the lab for a diagnosis, expecting a yes or no, but often end up with just a maybe…
Basic Principles of Lab diagnosis
Collecting and transporting specimens
Non-cultural diagnostic methods
Diagnosis of Bacterial Infection Patient Clinical diagnosis Haematology Biochemistry Non-microbiological investigations Radiology Sample Take the correct specimen Take the specimen correctly Label & package the specimen up correctly Appropriate transport & storage of specimen
A proper clinical assessment is essential for optimal use of laboratory services!
Collecting the correct specimen
Pernasal swabs for pertussis
Sputum , not saliva
Blood culture bottles, not clotted blood
Correctly timed Gentamicin assays
Pus, not swabs
Getting the specimen to the lab
Problems in delay or inappropriate storage
delay in diagnosis & treatment
Blood cultures directly into incubator
CSF straight to lab
Don't put an entire surgical specimen into formalin!
Send a portion to microbiology in a sterile container
Collecting the specimen correctly
Take an mid-stream urine
avoids contamination with perineal flora
Avoid bloody tap
Make the patient gag!
Avoid contamination with skin organisms
Specimens & Infection Control
Please be considerate to lab staff!!
Label hazardous specimens
Don't send specimens to the lab without proper packing
Leaking or blood-stained specimens are not acceptable!!!
Factors limiting usefulness of bacteriological investigations
e.g. saliva instead of sputum
delay in transport / inappropriate storage
overgrowth by contaminants
e.g. blood cultures
insufficient sample / sampling error
e.g.in mycobacterial disease
Animal/patient has received antibiotics
Diagnosis of Bacterial Infection culture on plates or in broth identification by biochemical or serological tests on pure growth from single colony microscopy Decolorise Counterstain Stain unstained or stained with e.g. Gram stain sensitivities Serodiagnosis DNA technologies by disc diffusion methods, breakpoints or MICs
Microscopy Unstained preparations
“ Wet prep”
Dark-ground illumination for syphilis
Microscopy Stained preparations
Direct, e.g. auramine
Culture of Bacteria
For safe long-term culture, e.g. Lowenstein-Jensen media for TB
Liquid media (broth)
For enrichment or maximum sensitivity
Advantages of Solid Media
isolation of single clonal colonies
get bacterium in pure culture
identify by colonial morphology
quantification by colony-forming units
Identification of Bacteria
Non-cultural diagnostic methods
e.g. latex agglutination
e. g. agglutination tests, complement fixation tests, indirect immunofluorescence
Polymerase Chain Reaction
on solid media
disc diffusion technique
in liquid media
minimum inhibitory concentration (MIC) test
I think it’s time for a short Break!! Oooh yeah!!!
Bacterial cultivation Part 1a Selective /differential media
Form 8 groups of about 8 students
Each group will have 1 type of specimen
Demo selective and differentiation plates
Streaking bacteria on differentiation plates
One group report after each practical MUST be submitted
The process of growing microorganisms in culture by taking bacteria from the infection site (in vivo or environment) and grow them in artificial environment in the laboratory (in vitro).