The importance of Microbiological culture and sensitivity testing and its advantages for Health care professionals

1. The Importance Of
Culture
Dr Abhijeet Mane
Assistant Professor
Dept. of Microbiology
Bharati Vidyapeeth Deemed University Medical College
Pune

2. Historical Aspect
 Medical Microbiology – study of microbes that infect
humans, the diseases they cause and their diagnosis,
prevention and treatment.
 Girolamo Fracastoro (1546) – infectious diseases
transmitted by – direct contact, fomites and through
air.
 Antony van Leeuwenhoek (1675) – first microscope
 Oliver W Holmes (1843) & Ignaz Semmelweiss
(1847) – puerperal sepsis is contagious.
 John Snow (1854) – Cholera transmitted by
contaminated water from water pump.

3.  Louis Pasteur (1822-1895) – Father of Microbiology
 Joseph Lister (1827-1912) – Father of antiseptic
surgery
 Robert Koch (1843-1910) – isolated bacteria causing
anthrax, tuberculosis & cholera
 Paul Ehrlich (1854-1915) – earliest pioneers in field
of antimicrobial chemotherapy. Therapy for syphilis.
 Sir Alexander Fleming – Penicillin; discovery led to
formulation of other antimicrobials
Historical Aspect (contd.)

4. Sir Alexander Fleming

6. Drug Resistance
 Beta lactamases – major defense of GNB
 ESBLs - resistant to broad spectrum
cephalosporins, aztreonam, extended spectrum
penicillin.
 Resistance co-transferred by plasmids
 Increase in Vancomycin Resistant Enterococci

7. Emerging infections in India
 MDR & Nalidixic acid resistant Salmonella
typhi
 Vibrio cholerae 0139
 Plague
 Acinetobacter baumannii
 Low – level penicillin resistance Pneumococci
 Meningococcal meningitis

8. Factors influencing transmission and
spread
 Inadequate surveillance, preventive and control
measures
 Lack of health care facilities in rural areas
 Socioeconomic factors
 Inadequate & contaminated water supply, unhygienic
sanitation
 Unavailability of drugs & non compliance of patients
 Ineffective health education or lack of health
education

9. Why Bacterial Culture and
Sensitivity is necessary?
 Selective isolation of pathogenic organism
 Antibiotic Sensitivity testing
 Controls indiscriminate use of antibiotics
 Assist clinicians
 Reveals changing trends in local isolates
 Helps local pattern of antibiotic prescribing
 Epidemiological use

10. SPECIMEN SUSPECTED
DISEASE
LABORATORY
PROCEDURE
POSITIVE
FINDINGS
1. Throat culture (swab) Diphtheria Gram’s stain Delicate pleomorphic
gram-positive bacilli in
Chinese letter pattern
Methylene blue stain Light blue staining bacilli
with prominent
metachromatic granules
Acute streptococcal
pharyngitis
Direct fluorescent antibody
technique (after 4-6 hrs
incubation in Todd-Hewitt
broth)
Fluorescent cocci in
chains; use positive and
negative controls with
each stain
2. Sputum
Transtracheal aspirates
Bronchial washings
Bacterial pneumonia Gram’s stain Variety of bacterial types;
Streptococcus pneumoniae
with capsules, particularly
diagnostic
Tuberculosis
Pulmonary mycosis
Acid fast stain
Gram’s stain, Wright-
Giemsa’s stain or
Calcoflour white
Gram-Weigert stain
Acid fast bacilli
Budding yeasts,
pseudohyphae, true
hyphae, or fruiting bodies
Diagnosis of Infectious Disease by direct examination of culture specimens

11. 4. Cutaneous wounds or
purulent drainage from
subcutaneous sinuses
Bacterial cellulitis Gram’s stain Variety of bacterial types;
suspect anaerobic species
Gas gangrene
(myonecrosis)
Gram’s stain GPB suggesting
Clostridium perfringens;
spores usually not seen
Actinomycotic mycetoma Direct saline mount
Gram’s stain or modified
acid fast stain
“ sulfur granules “
Delicate, branching gram-
positive filaments;
Nocardia species may be
weakly acid fast
Eumycotic mycetoma Direct saline mount
Gram’s stain or
lactophenol cotton blue
mount
White, grayish or black
grains
True hyphae with focal
swellings or
chlamydospores
5. Urine Yeast infection Gram’s stain or Wright-
Giemsa stain
pseudohyphae, or budding
yeasts
Bacterial infection Gram’s stain Variety of bacterial types

12. 6. Cerebrospinal fluid Bacterial meningitis Gram’s stain Small gram negative
pleomorphic bacilli
(Haemophilus species)
Gram negative diplococci
(Neisseria meningitidis)
Methylene blue stain Gram positive diplococci
(Streptococcus
pneumoniae)
Acridine orange stain Bacterial forms that glow
brilliant orange under
ultraviolet illumination
Pneumococcal meningitis Quellung reaction (type
specific antisera)
Swelling and ground glass
appearance of bacterial
capsules
Cryptococcal meningitis India ink or nigrosin
mount
Encapsulated yeast cells
with buds attached by thin
thread
7. Eye Purulent conjunctivitis Gram’s stain Variety of bacterial types
Trachoma Giemsa stain of corneal
scrapings
Intracellular perinuclear
inclusion clusters

13. 8. Purulent urethral
discharge
Gonorrhea Gram’s stain Intracellular gram negative
diplococci
Chlamydial infection Direct fluorescent antibody
stain of smear
Elementary bodies
9. Purulent vaginal
discharge
Yeast infection Direct mount or
Gram’s stain
pseudohyphae or budding
yeasts
Trichomonas infection Direct mount Flagellates with darting
motility
Gardnerella vaginalis Pap stain or Gram’s stain
Measure pH of vaginal
secretions
“ clue cells “ or pH of
vaginal secretions > 5.5
10. Penile or vulvar ulcer
(chancre)
Primary syphilis Dark field mount of
chancre secretions
Tightly coiled motile
spirochetes
Chancroid Gram’s stain of ulcer
secretions or aspirate of
inguinal bubo
Intracellular and
extracellular small GNB

14. 11. Feces Purulent enterocolitis Gram’s stain Neutrophils and
aggregates of
staphylococci
Cholera Direct mount of alkaline
peptone water enrichment
Bacilli with characteristic
darting motility; no
neutrophils
Parasitic disease Direct saline or iodine
mounts
Examine purged
specimens
Adult parasites or parasite
fragments; protozoa or ova
12. Skin scrapings, nail
fragments, or plucked hair
Dermatophytosis 10% KOH mount Delicate hyphae or
clusters of spores
Tinea versicolor 10% KOH mount or
Lactophenol cotton blue
mount
Hyphae and spores
resembling spaghetti and
meatballs
13. Blood Relapsing fever (Borrelia) Wright’s or Giemsa stain
Dark field examination
Spirochetes with typical
morphology
Blood parasites; malaria;
trypanosomiasis; filariasis
Wright’s or Giemsa stain
Direct examination of
anticoagulated blood for
the presence of
microfilaria
Intracellular parasites
(malaria, babesia)
Extracellular forms;
trypanosomes or
microfilaria

15. Please send..
 Urine – Wide mouthed, sterile. Midstream.
 Pus – Frank pus preferred. If not, swab.
 Respiratory samples – Sterile, wide mouthed.
 Blood – Bactec bottles
 CSF – Room temperature

16. Please avoid…
 Sending sample in formalin
 24 hour sputum / urine collections
 Single swab – multiple requests
 Improper, unsterile, obviously contaminated
container
 Leaky container

17. The Diagnostic Cycle
 Pre analytic phase
 Analytic phase
 Post analytic phase

18. Patient consults physician
with signs/symptoms of
infectious disease
THE DIAGNOSTIC CYCLE
Physician examines patient
& makes a tentative clinical
diagnosis
Appropriate specimen(s)
is/are collected for culture.
All containers are properly
labeled.
Written orders are transcribed
to a laboratory request form.
Form & specimen are
transported to the laboratory
Upon receipt by the lab, data
from the request form is
entered into a computer file
or log book
Physician interprets
reports & institutes proper
therapy
Final culture report is prepared
& sent to the physicians office,
clinic or hospital
Subcultures are examined,
& results of identification
systems are examined
After incubation, cultures are
examined. Definitive identification
systems are set up
Specimens are processed.
Culture media are selected,
inoculated, & incubated.
Preliminary reports may
or may not be issued
Presumptive reports
may or may not be issued
Specimen is directly examined.
Microscopic mounts, smears, &
stains may or may not be set up
Pre-analytical
Post-analytical
Analytical

19. Empiric choice for OPD patients
Suspected clinical
diagnosis
Likely etiology Drugs of choice
Erysipelas, impetigo, cellulitis Grp A Streptococcus Phenoxymethyl penicillin, orally
Furuncle S. Aureus Dicloxacillin
Pharyngitis Grp A Strep Phenoxymethyl penicillin, orally
Otitis media Pneumococci, H.influenzae, Amoxicillin
Acute sinusitis Pneumococci, H.influenzae, Amox or TMP-SMZ
Aspiration pneumonia Mixed oropharyngeal flora
including anaerobes
Clindamycin
Pneumonia Pneumococci, Atypical bacteria Doxycycline, clarithromycin,
azithromycin
Cystitis E.coli, K.pneumoniae, Proteus
spp
FQ, nitrofurantoin
Gastroenteritis Shigella (TMP-SMZ, Amp, Cipro), Campylobacter(Azithro or Cipro),
E.histolytica (Metro)
Pelvic inflammatory disease N.gonorrhea, C.trachomatis,
anaerobes
Ofloxacin plus Metronidazole

20. Thank you !!!