Conventional Diagnosis of Bacterial Infections: Macroscopy, Microscopy, Culture, Biochemical Tests, and Antibiotic Susceptibility

Table of Contents

Macroscopy of Clinical Specimens:

Sputum Examination

Description of sputum: Purulent, Mucopurulent, Mucoid, Mucosalivary or bloody

Stool Macroscopy

Color of the specimen, consistency, presence of blood, mucus or pus.
Unformed, containing pus and mucus mixed with blood: Enteroinvasive E. coli (EIEC) dysentery Campylobacter enteritis, Shigellosis
Bloody diarrhea (without pus cells): Enterohemorrhagic E. coli (EHEC) 0157 infection
Watery stools: Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC) diarrhea
Rice water stools: Cholera
Unformed or watery and sometimes with blood, mucus, and pus: Salmonella infection
Normal faeces: Appear brown and formed or semi formed.

Urine Examination

Color of specimen, whether it is clear or cloudy
Cloudy: Bacterial urinary infection.
Depending on concentration, freshly discharged urine is often clear and pale yellow to yellow.

Cerebrospinal fluid (CSF)

Clear, slightly turbid, cloudy or definitely purulent, contains blood, contains clots.
Normal CSF: Appears clear and colorless.
Pus: Description of the presence of granules

Microscopic Examination:

Sputum Microscopy

Gram smear using purulent part of the sputum to a glass slide and examination of the smear for pus cells and predominant bacteria (Streptococcus pneumonia, Staphylococcus aureus, Hemophilus influenza, Klebsiella pneumoniae).
Ziehl-Neelsen smear to detect AFB with decontamination-concentration

Pus, ulcer and skin specimen microscopy

Before utilizing the swab to create gram smears, the culture media should be inoculated.

Examination of the smear for bacteria among the pus cells: S. aureus, Streptococcus pyogenes or other beta-haemolytic streptococci, anaerobic streptococci, or enterococci, Proteus species, E. coli or other coliforms, Pseusomonas aeruginosa or Bacteroides species, Clostridium perfringens or Bacillus anthracis
Additional Ziehl-Neelsen if Mycobacterium tuberculosis suspected
Examination by dark-field microscopy to detect treponemes

Feces microscopy

Faecal leucocytes: inflammation of the large intestine.
Mononuclear cells: mainly in typhoid.
Inflammatory diarrhoeal disease: Shigella species, Campylobacter species, Salmonella (non-typhoid serovars)
Basic fuchsin smear to detect campylobacters: if unformed and, or, contains mucus, pus, or blood.
Motility test and Gram stained smear when cholera is suspected.
Darting motility using dark-field microscopy.

Urine microscopy

Wet preparation to detect: significant pyuria, red cells, casts, bacteria
Wet mount uses sediment of centrifuged urine.
Examination of a Gram stained smear: when bacteria and, or white cells are seen in the wet preparation using urine sediment.

Cerebrospinal Fluid (CSF) Microscopy

Purulent or cloudy CSF

Pyogenic meningitis so tests the CSF for Gram-stained smear for bacteria and pus cells. Culture the CSF.

Slightly cloudy or clear CSF

Performing a cell count.
When cells predominantly pus cells: Examination of a Gram-stained smear for bacteria
Culture of CSF
Examination of the gram smear microscopically for pus cells and bacteria.
Supernatant for biochemical test

Upper Respiratory Tract Microscopy

For mouth and throat specimen: Gram smear
Albert stained smear when diphtheria is suspected

Blood Microscopy

Gram smear: To detect Gram positive and Gram-negative bacteria, particularly when the patient is an infant or young child.
Ziehl-Neelsen smear to detect AFB when the patient has AIDS or suspected HIV Disease

Culture Techniques:

It is a gold standard method.
When microscopy of the specimen demonstrates an acceptable quality, it is subjected to culture.

Media for Primary Isolation of Microorganisms

Enriched Blood Agar: For the primary recovery of most commonly encountered microorganisms.

Chocolate Agar: Neisseria gonorrhoeae and Haemophilus influenzae.

Inhibitory media

Modified Thayer-Martin: pathogenic Neisseria species
Loeffler Medium and Tellurite Medium: For inoculation of nasopharyngeal cultures for recovery of Corynebacterium diphtheriae.

Enteric Media

MacConkeyAgar, DeoxycholateAgar Differential medium: for the recovery of Gram-negative bacilli. Most Gram-positive bacteria are inhibited.
Salmonella-Shigella Agar Xylose- Lysine-Desoxycholate (XLD) Agar: Media more selective: Salmonella or Shigella from feces or other body fluids or secretions.
Selenite Broth, or Tetrathionate Broth: Enrichment broths for concentration of pathogenic Salmonella or Shigella

Supplemental Primary Culture Media for Recovery of Special Fastidious Organisms

Lowenstein-Jensen (LJ) Agar: Primary recovery of mycobacteria.
Mannitol Salt Agar: For the isolation of staphylococci.
Thiosulfate-Citrate-Bile Salts-Sucrose Agar (TCBS): Primary recovery of Vibrio cholerae from fecal or other contaminated material.
Skirrow’s: Isolation of Campylobacter.
Use of Columbia agar: for culturing blood but organisms such as S. pneumonia and Neisseria meningitidis no growth shown
Thioglycollate broth medium: to isolate strict anaerobes should an anaerobic infection be suspected
Subculture on blood agar, chocolate agar, and MacConkey agar

SPECIMEN	MEDIA	POSSIBLE MICROORGANISM
Respiratory	Blood Agar, Chocolate Agar, Lowenstein Media	Mycobacterium tuberculosis, Streptococcus pneumonia, Streptococcus pyogenes, Haemophilus influenza, Staphylococcus aureus, E. coli, Proteus, Klebsiella, Pseudomonas
Urine	CLED	Staphylococcu saprophyticus, Escherichia coli, Proteus species, Haemolytic streptococci, Pseudomonas aeruginosa Klebsiella strains
Feces	Mac Conkey Agar, Xylose Lysine Deoxycholate, Deoxycholate Agar, Selenite F broth, Skirrow’s media, Cooked meat media	Clostridium perfringen,,s Shigella species Salmonella serovars, Clostridium difficile, Campylobacter species, Staphylococcus aureu,s Escherichia coli, Vibrio cholerae
Genital( Gonococcal)	Modified Thayer Martin Agar	Neisseria gonorrhoeae
Cerebrospinal fluid	Blood Agar, Chocolate Agar, Mac Conkey Agar	Neisseria meningitides, Haemophilus influenzae type b, Escherichia coli, Salmonella serovars, Streptococcus pneumoniae
Pus, Ulcer, Skin specimen	Blood Agar, Mac Conkey Agar	Staphylococcus aureus, Pseudonomas aeruginosa, Proteus species, Enterococcus species, Anaerobic streptococci, Bacteriodes species, Clostridium perfringens

Biochemical Characterization Tests:

Beta-glucuronidase to identify E. coli
Bile solubility: To differentiate S. pneumoniae from other alpha-haemolytic streptococci
Catalase: To differentiate staphylococci from streptococci
Citrate utilization: To differentiate enterobacteria
Coagulase: To identify S. aureus
Dnase: To help identify S. aureus
Indole: To differentiate Gram negative rods
Litmus milk: To help identify clostridia
Lysine decarboxylase: To assist in the identification of Salmonella and Shigella
Urease: To help identify Proteus, Helicobacter pylori

Antibiotic Susceptibility Testing (AST):

Should be performed only when cultural growth of a pathogen is significant.
Hemophilus influenzae strains should be tested for betalactamase production and susceptibility to ampicillin, tetracycline, and co-trimoxazole (sputum)
S. pyogenes and Corynebacterium diphtheria: Benzylpenicillin and erythromycin (antibiotics of choice to treat both types of infection) (throat n mouth)
Problems are often encountered when testing the susceptibility of staphylococci: oxacillin disc (1mg) (Wounds)
For S. aureus, enterobacteria and non-fermentative Gram-negative rods, only routinely used antibiotics should be tested
Susceptibility test more important on cultures obtained from patients who are hospitalized or have a history of recurring UTI.
Strains of S. pneumoniae should be tested on blood agar for susceptibility to penicillin, tetracycline, and erythromycin (sputum)