Identifying Microbes

Part of Germ Theory

Methods for characterizing and identifying microorganisms using microscopy, staining, culture, and biological tests.

Why This Matters

Knowing that a patient has “an infection” is the beginning, not the end, of diagnosis. Knowing which organism is causing the infection — or which class of organisms — guides treatment, isolation decisions, and prognosis. A wound infected with Staphylococcus responds to different interventions than one infected with Clostridium (tetanus/gas gangrene). Cholera and typhoid both cause severe diarrhea but require different responses and have different transmission routes.

Modern clinical microbiology relies on sophisticated equipment — automated culture systems, DNA sequencing, mass spectrometry. But the foundational techniques developed by Koch and his contemporaries in the 1870s-1890s require only a microscope, staining chemicals, some glassware, and biological media that can be made from simple ingredients. These techniques allow meaningful differentiation between pathogen types and can guide clinical decisions significantly better than examination alone.

For a rebuilding community with access to a functioning microscope, basic microbiology is achievable. It requires patience and practice, but the skills are learnable and the information gained is clinically valuable.

Step 1: Specimen Collection

The value of any identification procedure depends entirely on the quality of the specimen. Poor specimens produce misleading results.

From wounds: Take specimens from the deep edge of a wound, not the surface. The surface contains environmental contamination; the organisms causing the infection are at the advancing edge. Use a sterile swab or wire loop to collect material from the deepest accessible part of the wound.

From sputum: For respiratory infections, collect a deep cough sample — not saliva. Ask the patient to take a deep breath and cough forcefully from the chest. Collect sputum in a clean container. Early morning specimens before eating yield the highest organism counts.

From stool: For diarrheal illness, collect a small amount of fresh stool (not touching the toilet) in a clean container. Process promptly — many stool pathogens do not survive well outside the host.

From blood: Blood cultures require sterile collection technique — skin is cleaned with iodine, blood is drawn with a sterile needle, and inoculated into sterile culture broth. This is a more demanding procedure but extremely valuable when septicemia (bloodstream infection) is suspected.

From urine: Collect midstream urine after cleaning the urethral opening — the first portion flushes the urethra, the midstream portion reflects what is in the bladder.

Step 2: Microscopic Examination

A direct smear provides a rapid preliminary view.

Wet mount:

• Place a small amount of specimen in a drop of clean water on a clean glass slide
• Cover with a coverslip
• Examine under microscope (100-400x)
• Allows visualization of protozoa (moving), yeasts, fungal hyphae, and occasionally bacteria
• Cannot distinguish between most bacteria — too small and unstained to characterize

Fixed smear for staining:

• Spread a thin film of specimen on a clean slide
• Allow to air dry completely
• Heat-fix by passing through a flame 3-5 times — kills organisms, adheres them to the glass
• Ready for staining

Step 3: Staining

Staining makes bacteria visible and differentiates them into categories. See the dedicated staining techniques article for full detail on the Gram stain procedure. In brief:

Gram stain results:

• Gram-positive organisms (purple/violet after staining): Staphylococcus, Streptococcus, Clostridium, Bacillus, Listeria, Pneumococcus
• Gram-negative organisms (pink/red after counterstaining): E. coli, Salmonella, Shigella, Vibrio, Klebsiella, Pseudomonas

Combined with morphology (cocci, bacilli, spirals) and arrangement (clusters, chains, pairs), Gram stain results narrow the differential diagnosis substantially.

Acid-fast stain (for tuberculosis): Mycobacterium tuberculosis has a waxy cell wall that resists Gram staining but retains red carbol fuchsin dye even after acid-alcohol washing (“acid-fast”). A positive acid-fast smear from sputum is strong presumptive evidence of pulmonary tuberculosis. Ziehl-Neelsen staining (carbol fuchsin + acid-alcohol decolorizer + methylene blue counterstain) is the standard method.

Step 4: Culture

Culture grows organisms in sufficient quantity for further testing and identification. It requires:

Nutrient broth: Beef extract or bone broth (boiled bone marrow) in water, clarified by filtering through cloth, provides a nutrient medium. Add 0.5% salt (5 g/L). Sterilize by pressure cooking (121°C, 15 min). Inoculate from specimen and incubate at body temperature (37°C).

Solid agar: Natural agar (from seaweed) or gelatin (from bones) can solidify nutrient broth into a gel. Gelatin melts above 25°C and is less ideal; agar remains solid to 65°C. Pour molten agar into flat dishes (Petri dishes) or test tubes. Allow to solidify, inoculate on the surface, incubate.

Selective media: Adding specific chemicals can inhibit some organisms while allowing others to grow. Salt agar (7.5% NaCl) allows Staphylococcus (salt-tolerant) to grow while inhibiting many other organisms. This kind of selective pressure is achievable without sophisticated reagents.

Observing growth:

• Growth in liquid broth: turbidity (cloudiness) usually visible within 24-48 hours
• Growth on solid media: colonies visible in 24-72 hours — each colony is a clone derived from a single organism
• Colony morphology (size, shape, color, texture) varies by species and provides identification clues

Colony characteristics:

• Staphylococcus aureus: golden-yellow, round, opaque colonies
• Streptococcus: small, grey, translucent colonies
• E. coli: large, flat, grey, often with metallic sheen
• Pseudomonas: may produce blue-green pigment (pyocyanin) visible in colony and surrounding agar

Step 5: Biochemical Tests

Simple biochemical tests further differentiate organisms.

Catalase test:

• Apply a drop of 3% hydrogen peroxide to a colony
• Bubbling (oxygen production) = catalase-positive = Staphylococcus (catalase-positive gram-positive cocci)
• No bubbling = catalase-negative = Streptococcus
• This simple test rapidly distinguishes between the two most common gram-positive cocci

Oxidase test:

• Not easily performed without commercial reagent, but oxidase-positive organisms (Pseudomonas, Vibrio) can sometimes be identified by colony characteristics combined with other tests

Motility:

• Inoculate a stab into semi-solid agar (0.3-0.5% agar concentration)
• Motile organisms spread outward from the inoculation line; non-motile stay in the line
• Many pathogens (E. coli, Salmonella, Vibrio) are motile; Klebsiella is non-motile

Hemolysis on blood agar:

• Blood agar made by adding 5% defibrinated sheep blood to sterile agar
• Alpha hemolysis: partial clearing, greenish zone (Streptococcus pneumoniae)
• Beta hemolysis: complete clear zone (Streptococcus pyogenes, Staphylococcus aureus)
• Gamma hemolysis: no clearing (many organisms)

Interpreting Results Clinically

Identification results must be interpreted in clinical context. A wound with gram-positive cocci in clusters (Staphylococcus) warrants different management than one with gram-negative rods with gas production (Clostridium perfringens, anaerobes).

A reasonable field microbiology workflow for a serious infection:

1. Collect specimen with sterile technique
2. Gram stain — determines gram reaction and morphology in 30 minutes
3. Culture — observe growth at 24 and 48 hours
4. Apply basic biochemical tests to colonies
5. Integrate findings with clinical presentation

This process cannot replace modern diagnostic microbiology, but it can distinguish between the broad categories of infection that most practically affect treatment decisions: gram-positive vs. gram-negative, aerobe vs. anaerobe, bacteria vs. fungus vs. parasite. Each of these distinctions has practical treatment implications even in a resource-limited setting.