Manual for Practical Classes in Special Microbiology: Study Guide

Laboratory Practice and Safety Standards

• Operational Safety in a Bacteriological Laboratory: Mastery of laboratory practice rules is mandatory to protect against pathogenic microorganisms and fire hazards.
• Behavioral Standards: High standards of behavior, a sense of responsibility, and carefulness are essential for academic integrity and personal safety.
• Handling Living Cultures: All cultures, including non-pathogenic ones, require respect and proper technique to prepare for future work with highly pathogenic bacteria.
• Aseptic Environment: A clean, well-ordered environment guarantees aseptic technique, reliable results, and better learning.
• Contamination Prevention: Defined as the process of environmental microorganisms entering specimens or cultures and interfering with results.
• Basic Rules and Precautions:
  • Outerwear: Removal of street coats, jackets, and other outer-wear before entry to prevent fire or contamination.
  • Attire: Mandatory clean medical white overall and medical cap to protect skin and street clothes.
  • Footwear: Mandatory use of second (indoor) shoes or disposable shoe covers.
  • Hair: Long hair must be bound back and kept under the cap.
  • Hygiene: Hands must be washed with antiseptic soap or disinfectant before and after lab work.
  • Object Control: Fingers, pencils, and pens must be kept away from tubes, Petri dishes, and the mouth.
  • Prohibited Activities: No smoking, drinking, eating, or cosmetic application in the lab.
  • Conduct: No wandering, noise, or loud talking to prevent accidents and distraction.
  • Aseptic Technique: Work must be performed near a spirit burner; cultures are transferred using a sterile wire loop.
  • Loop Sterilization: Contaminated loops must be sterilized in the flame before resting; heating must be done gradually to avoid spattering bacteria.
  • Storage: Tubes must always be kept in tube racks.
  • Spillage: Immediate notification of the teacher for disinfection is required after any spillage.
  • Removal of Materials: No culture, glassware, media, or chemicals may be removed from the lab.
  • Waste Disposal: Solid waste (used glasses, pipettes) goes into special vessels with disinfectant. Dyes are discarded in specific sinks in separate rooms. No contaminated materials in general sinks.
  • Workplace Order: The workplace must be left in perfect order after each session.

Lesson 17: Coccal Infections and Microbiological Diagnostics (Staphylococcus)

• Taxonomy and General Characteristics: Staphylococci are Gram-positive cocci arranged in grape-like clusters (Greek: staphyle, bunch of grapes). Members are non-flagellate, non-motile, non-spore-forming, and facultatively anaerobic. They produce catalase.
• Species of Medical Importance: More than one dozen species colonize humans; major ones include $S. aureus$, $S. epidermidis$, and $S. saprophyticus$. Currently, the genus includes $32$ species.
• Classification by Coagulase: Species are divided into two groups based on the ability to clot human or rabbit plasma. Coagulase-positive species include $S. aureus$, $S. intermedius$, $S. hyicus$, and $S. delphini$.
• Surface Structures: The cell wall features Protein A, unique for binding the Fc portion of $IgG$ molecules, leaving the Fab portion external. This is used in antigen detection systems.
• Virulence Factors of $S. aureus$:
  • Enzymes: Lecitinase, phosphatase, coagulase, hyaluronidase, fibrinolysin, and desoxyribonuclease.
  • Haemolysins: Four types (alpha, beta, gamma, delta). Alpha lysin is a cytotoxic, leucocidal, and dermonecrotic protein inactivated at $60^{\circ}\text{C}$.
  • Leucocidin (Panton-Valentine toxin): Composed of S (slow) and F (fast) components; damages polymorphonuclear leucocytes and macrophages.
  • Enterotoxins: Antigenically distinct proteins (A, B, $C1$, $C2$, $C3$, D, E, H, I). They are heat-stable (survive boiling) and act on neural receptors in the upper GI tract to stimulate vomiting.
  • Exfoliatins: Causes intercellular splitting of the epidermis between the stratum spinosum and stratum granulosum. Variants A and B exist.
  • Toxic Shock Syndrome Toxin-1 (TSST-1): A superantigen causing cytokine release, capillary leakage, hypotension, and shock.
  • Pyrogenic Toxin Superantigens (PTSAgs): Stimulate T cells and macrophages to release massive amounts of $TNF\text{-alpha}$ and $IL\text{-1}$.
• Epidemiology: The primary habitat of $S. aureus$ is the anterior nares (approx. $30\%$ of individuals). Most community infections are autoinfections. Hospital outbreaks often involve hands of personnel or heavy nasal/perineal carriage.
• Staphylococcal Food Poisoning: Result of bacterial multiplication in moist, rich foods (e.g., potato salad). Toxins persist even after boiling.
• Clinical Manifestations:
  • Primary Infections: Furuncles (boils in hair follicles/glands) and Carbuncles (deep subcutaneous abscesses causing bacteremia).
  • Chronic Furunculosis: Repeated attacks by the same strain, often associated with diabetes or immune defects; involves delayed-type hypersensitivity.
  • Impetigo: Bullous form caused by exfoliatin-producing strains.
  • Scalded Skin Syndrome: Erythema and intraepidermal desquamation; common in neonates and children under $5$ years.
• Laboratory Diagnosis: Gram-staining shows Gram-positive cocci in clusters. Growth on Blood agar, Mannitol-salt agar, or Egg yolk salt agar. Catalase and coagulase tests are definitive.
• Treatment: Penicillin-resistant strains ($80\text{-}90\%$) are common due to $\beta\text{-lactamases}$. Alternatives include methicillin, nafcillin, oxacillin, vancomycin, clindamycin, or erythromycin.

Lesson 18: Streptococcus spp. & Neisseria spp.

• Streptococcus General Features: Gram-positive, ovoid cocci in chains. Non-acid fast, non-motile, non-spore-forming. Catalase negative.
• Hemolysis Patterns on Blood Agar:
  • $\beta\text{-hemolysis}$: Clear zone of complete hemolysis.
  • $\alpha\text{-hemolysis}$: Hazy, incomplete green discoloration.
• Classification: Based on Rebecca Lancefield’s serogroups (A, B, C, etc.) targeting carbohydrate antigens in the cell wall.
• Group A Streptococci ($S. pyogenes$):
  • Virulence: M protein (fibrillar molecule, $80+$ serotypes), Streptolysin O (oxygen-labile, antigenic, forms basis of ASO test), and Streptolysin S.
  • Streptococcal Pyrogenic Exotoxins (SPEs): Associated with Scarlet Fever; act via superantigen mechanism.
  • Diseases: Pharyngitis ("strep throat"), Impetigo, Erysipelas (dermic infection), and Scarlet fever (strawberry tongue, sandpaper rash).
  • Sequelae: Acute Rheumatic Fever (ARF) and Acute Glomerulonephritis (AGN).
• Streptococcus pneumoniae (Pneumococci): Lancet-shaped, Gram-positive diplococci. All virulent strains possess a polysaccharide capsule ($90+$ serotypes).
  • Diagnostics: Bile solubility test (autolysin activation) and Optochin susceptibility differentiate them from viridans streptococci.
• Neisseria General Features: Gram-negative "kidney bean" shaped diplococci. Oxidase positive. Require carbon dioxide and enriched media (Chocolate agar).
• Neisseria meningitidis (Meningococcus): Has a polysaccharide capsule ($12$ serogroups: A, B, C, H, I, K, L, X, Y, Z, 29E, $W\text{-}135$).
  • Clinical: Acute purulent meningitis and Meningococcemia (characterized by petechial rash and disseminated intravascular coagulation/DIC).
  • Waterhouse–Friderichsen Syndrome: Bilateral hemorrhagic destruction of the adrenal glands during fulminant disease.
• Neisseria gonorrhoeae (Gonococcus): Primarily localized to mucosal surfaces. Possesses pili for attachment.
  • Clinical: Urethritis in men, cervical infection in women. Can lead to Pelvic Inflammatory Disease (PID), sterility, or ectopic pregnancy.
  • Asymptomatic Carriers: $50\%$ of infected women and a portion of men are asymptomatic but infectious.
  • Diagnosis: Direct Gram smear ($95\%$ sensitive in symptomatic men; $50\text{-}70\%$ in women). Culture on Martin-Lewis or Thayer-Martin agar (enriched selective chocolate agar).

Lesson 19: Mycobacterium spp. (Tuberculosis and Leprosy)

• General Characteristics: Slender, Gram-positive, acid-fast bacilli (AFB). Cell walls contain over $60\%$ mycolic acids (long-chain fatty acids) and Lipoarabinomannan (LAM).
• Mycobacterium tuberculosis:
  • Morphology: Straight or slightly curved bacilli. Stained by Ziehl-Neelsen (ZN) or fluorescent dyes (Auramine O, Rhodamine).
  • Cultivation: Lowenstein-Jensen (L-J) medium (eggs, asparagine, malachite green, glycerol). Slow growth: $2\text{-}8$ weeks.
  • Serpentine Cords: Virulent strains grow in cords (Preus’ rapid test) due to "cord factor" (trehalose linked to mycolic acids).
• Pathogenesis:
  • Delayed-type Hypersensitivity (DTH): Destroys nonactivated macrophages; detected by skin tests.
  • Cell-mediated Immunity (CMI): Activates macrophages to destroy bacilli.
  • Koch’s Phenomenon: Reaction in a previously infected animal showing accelerated, localized necrotic response to reinfection, indicating combined immunity and hypersensitivity.
  • Ghon Complex: Characteristic radiographic picture of a fibrosed/calcified primary site and hilar lymph nodes.
• Laboratory Diagnosis:
  • Microscopy: ZN stain or fluorescence.
  • Petroff’s Method: Sputum homogenization/concentration using $4\%$ sodium hydroxide.
  • Tuberculin Skin Test (PPD): Intradermal injection of purified protein derivative. Induration of $10\,\text{mm}$ or more indicates hyperergy (past or present infection).
• Treatment and Prophylaxis:
  • BCG Vaccine: Live attenuated strain of $M. bovis$. Administered intradermally at birth.
  • RNTCP (Revised National Tuberculosis Control Programme): Diagnosis based primarily on microscopy of two sputum samples. Sputum-negative cases are diagnosed via X-ray.
• Mycobacterium leprae: Obligate parasite of humans; causes leprosy.

Lesson 20: Corynebacterium spp.

• Corynebacterium diphtheriae: Gram-positive, pleomorphic, club-shaped bacilli. Feature metachromatic granules (Babes-Ernst or volutin granules) containing polymetaphosphates.
• Staining: Albert stain (green bacilli, bluish-black granules) or Neisser stain (yellow bacilli, brown granules).
• Arrangement: Chinese letter or cuneiform (V or L shapes) due to incomplete binary fission.
• Biochemicals: Ferments glucose/maltose (acid, no gas). PYZ-negative and urease-negative.
• Diphtheria Exotoxin: Produced by strains carrying the tox gene via lysogenic phage (beta phage).
  • Structure: Fragment A (enzymatic activity) and Fragment B (binding).
  • Mechanism: Inhibits protein synthesis by inactivating elongation factor $EF\text{-}2$ through ADPR binding.
• Clinical Signs: Pseudomembrane (grey-white, leathery exudate on tonsils/pharynx). Systemic effects include myocarditis, polyneuropathy, and adrenal failure.
• Diagnosis:
  • Loeffler’s Serum Slope: Rapid growth ($6\text{-}8$ hours).
  • Tellurite Blood Agar: Black/grey colonies due to tellurite reduction.
  • Elek's Gel Precipitation Test: In vitro immunodiffusion test for toxigenicity detection.
• Prophylaxis: Active immunization via DPT (Triple) vaccine at $6$ weeks. Passive immunization with antidiphtheric serum (ADS).

Lesson 21: Escherichia spp. & Shigella spp.

• Enterobacteriaceae Family: Gram-negative, facultative anaerobic bacilli. Glucose fermenting, oxidase negative, catalase positive (except $Sh. dysenteriae$ type $1$).
• Escherichia coli:
  • Antigens: $O$ (somatic, heat-stable LPS, $173$ types), $K$ (capsular, acidic polysaccharide, $103$ types), $H$ (flagellar, thermolabile, $75$ types), and $F$ (fimbrial).
  • Groups of Diarrheagenic $E. coli$:
    • EPEC (Enteropathogenic): Infant diarrhea; tight adherence to enterocytes.
    • ETEC (Enterotoxigenic): Traveler's diarrhea; produces LT (heat-labile, resembles cholera toxin) and ST (heat-stable).
    • EIEC (Enteroinvasive): Shigella-like dysentery; invades epithelial cells. Positive Sereny test (keratoconjunctivitis in guinea pigs).
    • EHEC (Enterohemorrhagic): Serotype $O157:H7$; produces Verotoxin; causes hemorrhagic colitis and Hemolytic Uremic Syndrome (HUS).
    • EAEC (Enteroaggregative): 'Stacked brick' formation on cells; produces $EAST\,1$ toxin.
• Shigella:
  • Classification: Subgroup A ($Sh. dysenteriae$ - mannitol negative), B ($Sh. flexneri$), C ($Sh. boydii$), D ($Sh. sonnei$ - late lactose fermenter).
  • Pathogenesis: Bacillary dysentery with low infective dose ($10\text{-}100$ bacilli). Virulence Marker Antigens (VMA) are plasmid-encoded.
  • Culture: DCA (Deoxycholate Citrate Agar), XLD, or SS agar. Shigella are pale (NLF); Salmonella are pale with black centers (H2S).

Lesson 22: Salmonella spp.

• General Characteristics: Gram-negative, motile (peritrichous flagella), H2S producers (except $S. paratyphi A$).
• Antigenic Structure (Kauffmann-White Scheme):
  • H (Flagellar): Phase $1$ and Phase $2$. Rapidly forms loose, fluffy clumps.
  • O (Somatic): LPS; forms compact granular clumps.
  • Vi (Surface): Enhances virulence by inhibiting phagocytosis. Found in $S. typhi$, $S. paratyphi C$, and $S. dublin$.
• Enteric Fever (Typhoid/Paratyphoid):
  • Pathogenesis Stages: 1. Digestive, 2. Mesenteric lymphadenitis, 3. Bacteremia (1st week), 4. Parenchymatic diffusion (gall bladder), 5. Inflammative-allergic (Peyer's patches/necrosis), 6. Excretory (stool/urine).
• Laboratory Diagnosis: Blood culture is positive in $90\%$ of cases in the first week. Clot culture is an alternative.
• Widal Test: Agglutination test for antibodies ($H$ and $O$). A four-fold rising titer or a single $O\,1:100$ / $H\,1:200$ suggests active infection.
• Prophylaxis: TAB vaccine (killed), Live oral ($Ty\,21a$), or Purified $Vi$ polysaccharide vaccine.

Lesson 23: Cholera ($Vibrio cholerae$)

• General Characteristics: Gram-negative, curved ("comma") rods. Actively motile via a polar flagellum ("darting motility"). Fish-in-stream appearance in mucous flakes. Oxidase positive.
• Classification:
  • $O1$ Serogroup: Includes Classical and El Tor biotypes. Subdivided into serotypes Ogawa, Inaba, and Hikojima.
  • $O139$ Serogroup: Bengal strain; emerged in $1992$.
  • $NAG$/Non-$O1$ Vibrios: Serogroups $O2\text{-}O138$. Similar to $V. cholerae$ but not agglutinated by $O1$ antiserum.
• Cholera Toxin (CT): Fragment A stimulates adenyl cyclase, converting ATP to $cAMP$. Massive concentration of $cAMP$ causes hypersecretion of water and electrolytes, resulting in "rice water stool."
• Diagnostics:
  • Media: Alkaline Peptone Water (enrichment), TCBS (yellow colonies), BSA (translucent colonies).
  • Darting Motility: Observed under dark field/phase contrast; inhibited by antiserum.
• Treatment: Immediate Oral Rehydration Therapy (ORT). Tetracycline is used secondarily.
• Prophylaxis: Killed parenteral or oral vaccines (BS-WC). Active immunization provides $50\text{-}60\%$ protection for $3\text{-}6$ months.

Questions & Discussion

• What is the differentiation of S. aureus from other species?
  • Differentiated based on the production of coagulase, fermentation of mannitol, and presence of lecitinase.
• What are MRSA and MRSE?
  • Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus epidermidis, signifying types of hospital-acquired or community multi-drug resistance.
• How do you distinguish C. diphtheriae from diphtheroids?
  • $C. diphtheriae$ have metachromatic granules, Chinese letter arrangement, and are toxigenic/urease negative. Diphtheroids are usually urease positive, lack granules, and arrange in palisades.
• What results confirm the etiology of a purulent postoperative complication?
  • Isolation of more than $1000\,\text{CFU}$ on a swab, presence of coagulase, and characteristic biochemical profiles.
• What is the clinical value of the PPD test?
  • It measures prevalence in a community, diagnoses active infection in children, and indicates successful BCG vaccination.
• Is a direct smear suitable for diagnosing diphtheria?
  • No, because it cannot define toxigenicity and is unreliable due to the presence of other organisms looking similar to $C. diphtheriae$ in the throat.