Topic 1. Morphology and principles of cultivating bacteria
Introduction
Bacteria are among the most important microorganisms responsible for ocular infections. Understanding their morphology and methods of cultivation is fundamental for optometry students, as accurate identification of bacterial species helps in diagnosis, treatment, and prevention of eye diseases. The eye, although protected by anatomical barriers and antimicrobial secretions like tears, is still vulnerable to bacterial infections such as conjunctivitis, keratitis, dacryocystitis, and endophthalmitis. Hence, a detailed study of bacterial morphology and their cultivation techniques provides the basis for ocular microbiology.
General Morphology of Bacteria
The term morphology refers to the external form, size, arrangement, and internal structures of bacteria. Bacteria are prokaryotic organisms (lacking a true nucleus) and measure between 0.2 to 2.0 micrometers in size. Morphological study is essential for classification and identification.
1. Bacterial Shapes
- Cocci (spherical) – e.g., Staphylococcus aureus (causes stye, bacterial conjunctivitis).
- Bacilli (rod-shaped) – e.g., Pseudomonas aeruginosa (associated with keratitis, particularly in contact lens users).
- Vibrios – comma-shaped bacteria.
- Spirilla – rigid spiral-shaped bacteria.
- Spirochetes – flexible spiral bacteria (e.g., Treponema pallidum in ocular syphilis).
- Actinomycetes – filamentous branching bacteria.
2. Bacterial Arrangement
- Diplococci – Cocci arranged in pairs (e.g., Neisseria gonorrhoeae, causing gonococcal conjunctivitis).
- Streptococci – Chains of cocci.
- Staphylococci – Grapelike clusters (common in eyelid and corneal infections).
- Tetrads and Sarcinae – Groups of four or cubical packets of eight cocci.
3. Structural Components of Bacteria
The internal and external structures of bacteria are essential for understanding how they survive, cause disease, and respond to treatment. In ocular microbiology, many eye infections are explained by the presence of specialized structures such as capsules, pili, or toxins. Below is a detailed overview:
a) Cell Wall
The bacterial cell wall is the rigid outer covering that provides shape, prevents osmotic lysis, and plays a critical role in Gram staining. It is mainly composed of peptidoglycan (murein), which is a network of sugars (N-acetyl glucosamine and N-acetyl muramic acid) cross-linked by peptides.
- Gram-positive bacteria – Have a thick peptidoglycan layer and teichoic acids. They appear purple after Gram staining. Example: Staphylococcus aureus, a major cause of blepharitis and keratitis.
- Gram-negative bacteria – Have a thinner peptidoglycan layer but possess an outer membrane containing lipopolysaccharide (LPS), which acts as an endotoxin and contributes to virulence. They appear pink/red after Gram staining. Example: Pseudomonas aeruginosa, known for aggressive corneal ulcers in contact lens wearers.
The difference between Gram-positive and Gram-negative bacteria is not just diagnostic but also influences the choice of antibiotics. For example, Gram-negative organisms are generally more resistant due to the protective outer membrane.
b) Gram Staining Process
Gram staining is a fundamental laboratory test to classify bacteria. The steps are:
- Primary stain – Crystal violet stains all bacteria.
- Mordant – Iodine is added, forming a dye-iodine complex.
- Decolorizer – Alcohol or acetone removes the stain from Gram-negative bacteria but not from Gram-positive due to thick peptidoglycan.
- Counterstain – Safranin stains Gram-negative bacteria pink/red.
This simple test is crucial in ocular microbiology labs for rapid differentiation. For example, corneal scraping examined under Gram stain can immediately point towards bacterial type before culture results are available.
c) Capsule
The capsule is a polysaccharide (sometimes polypeptide) layer surrounding the cell wall. It is not essential for survival but significantly increases virulence by protecting bacteria from phagocytosis. Capsules also contribute to biofilm formation on contact lenses and intraocular devices.
- Klebsiella pneumoniae – Known for thick capsule, associated with conjunctivitis and endophthalmitis.
- Pseudomonas aeruginosa – Forms biofilms on contact lenses, making infections more severe and resistant to treatment.
d) Flagella
Flagella are long, whip-like structures responsible for bacterial motility. Motile bacteria can reach deeper ocular tissues and spread rapidly. Flagella consist of three parts: filament, hook, and basal body. They are antigenic and classified as H-antigens.
- Monotrichous – Single flagellum (e.g., Pseudomonas).
- Lophotrichous – Tufts of flagella at one end.
- Amphitrichous – One flagellum at both ends.
- Peritrichous – Flagella all over the surface (e.g., E. coli).
Flagella allow bacteria to move towards favorable environments and away from host defenses, thereby aiding ocular colonization.
e) Pili and Fimbriae
Pili (singular: pilus) are short, hair-like projections used mainly for attachment to host tissues. They are critical in establishing infections on the conjunctival and corneal epithelium. There are two main types:
- Common pili (fimbriae) – Mediate adhesion to epithelial cells, enabling colonization. Example: Neisseria gonorrhoeae pili facilitate attachment to conjunctival epithelium in gonococcal conjunctivitis.
- Sex pili – Involved in bacterial conjugation and transfer of genetic material, often carrying antibiotic resistance genes.
f) Spores
Some Gram-positive bacteria (e.g., Bacillus, Clostridium) form endospores, which are highly resistant dormant structures. Spores can withstand heat, desiccation, and disinfectants. Though ocular spore-forming infections are rare, spore biology is important for sterilization protocols in ophthalmic practice to prevent contamination of surgical instruments.
g) Cytoplasmic Components
- Nucleoid – Contains the single circular bacterial chromosome.
- Plasmids – Small DNA molecules carrying antibiotic resistance genes (e.g., multidrug-resistant ocular pathogens).
- Ribosomes – Sites of protein synthesis, target for many antibiotics such as tetracyclines and aminoglycosides.
- Inclusion bodies – Storage granules for nutrients.
By studying these structures, students can understand why certain bacteria are highly infectious, why some resist antibiotics, and how laboratory tests like Gram staining or culture provide rapid insights into bacterial identity.
Principles of Bacterial Cultivation
Cultivation of bacteria refers to growing them in controlled environments outside the human body. This is essential for studying their morphology, physiology, biochemistry, and pathogenicity. Cultivation also enables antibiotic sensitivity testing, which guides treatment.
1. Basic Requirements for Bacterial Growth
- Nutrients – Carbon, nitrogen, vitamins, and minerals.
- Moisture – Required for metabolic activities.
- Temperature – Most pathogenic bacteria are mesophiles (optimal growth at 35–37°C, similar to human body temperature).
- pH – Most grow optimally at pH 7.2–7.4.
- Oxygen requirement –
- Aerobic – Require oxygen (e.g., Pseudomonas).
- Anaerobic – Grow without oxygen (e.g., Clostridium species).
- Facultative anaerobes – Can grow with or without oxygen (e.g., E. coli).
2. Types of Culture Media
Bacteria require specific media for growth. Media can be classified as:
- Simple media – Nutrient broth, nutrient agar; supports non-fastidious bacteria.
- Enriched media – Blood agar, chocolate agar; supports fastidious organisms (e.g., Haemophilus influenzae causing conjunctivitis).
- Select ive media – Contains inhibitors to suppress unwanted organisms (e.g., Thayer-Martin medium for Neisseria gonorrhoeae).
- Differential media – Helps differentiate bacteria based on biochemical reactions (e.g., MacConkey agar distinguishing lactose fermenters from non-fermenters).
- Transport media – Maintains viability during transfer (e.g., Stuart’s medium for conjunctival swabs).
3. Cultivation Methods
- Streak culture – For isolation of pure colonies.
- Stab culture – Used to test motility and oxygen requirements.
- Pour plate method – For viable count of bacteria.
- Broth culture – For obtaining bacterial growth in liquid medium.
4. Growth Curve of Bacteria
When cultivated in a laboratory, bacteria follow a predictable growth curve:
- Lag phase – Adaptation, no cell division.
- Log (exponential) phase – Rapid multiplication.
- Stationary phase – Nutrient depletion, growth slows.
- Death phase – Decline in viable cells.
Topic-2 Sterilization and disinfections used in laboratory and hospital practice
Introduction
In ocular microbiology and ophthalmic practice, the control of microorganisms is of utmost importance. The eye is highly sensitive to infection, especially following surgery, trauma, or use of contact lenses. Therefore, understanding the principles of sterilization and disinfection is essential for both laboratory and hospital environments. Sterilization ensures complete elimination of all forms of microbial life, while disinfection reduces harmful organisms to a safe level. Both play a critical role in preventing postoperative endophthalmitis, keratitis, and hospital-acquired ocular infections.
Definitions
- Sterilization – The complete removal or destruction of all forms of microorganisms including bacteria, spores, fungi, and viruses. A sterile item is totally free of all living organisms.
- Disinfection – The process of eliminating most pathogenic microorganisms (except bacterial spores) from inanimate objects or surfaces.
- Antisepsis – The application of chemical agents on living tissues to inhibit or kill microbes (e.g., povidone iodine used on conjunctiva before surgery).
- Sanitization – Reduction of microbial population to safe levels, often applied in general cleaning.
Principles of Sterilization and Disinfection
The effectiveness of sterilization and disinfection depends on several factors:
- Nature of the microorganism (spores are hardest to kill).
- Number of organisms present (greater load requires longer exposure).
- Concentration and potency of disinfecting agent.
- Duration of exposure.
- Presence of organic matter (blood, tears, pus) which can protect microbes.
- Type of material being sterilized (heat-sensitive vs. heat-stable).
Methods of Sterilization
Sterilization can be broadly divided into physical and chemical methods.
1. Physical Methods
a) Heat Sterilization
Heat is the most reliable and widely used method.
- Moist heat (Autoclaving) –
- Uses steam under pressure (121°C at 15 psi for 15–20 minutes).
- Destroys all microorganisms including spores.
- Used for surgical instruments, culture media, dressings.
- In ophthalmology: sterilization of instruments like forceps, scissors, specula, and reusable syringes.
- Boiling – 100°C for 10–30 minutes kills most bacteria but not spores; not a reliable method for ophthalmic instruments.
- Pasteurization – 60–70°C for 30 minutes; used mainly for milk, not relevant for hospital sterilization.
- Dry heat (Hot air oven) – 160–180°C for 1–2 hours.
- Used for glassware, Petri dishes, oils, powders.
- Common in microbiology labs for sterilizing glass slides used in Gram stain and other ocular smear studies.
b) Filtration
Filtration removes microorganisms without killing them. Membrane filters (0.22 µm) are used for heat-sensitive solutions such as antibiotic eye drops and intraocular irrigating fluids.
c) Radiation
- Ultraviolet (UV) radiation – Used to disinfect air and surfaces in operation theatres and laboratories.
- Ionizing radiation (Gamma rays) – Used industrially to sterilize disposable ophthalmic syringes, intraocular lenses, and sutures.
d) Chemical Sterilization (Gas)
Certain chemicals in gaseous form achieve sterilization:
- Ethylene oxide (ETO) – Used for heat- and moisture-sensitive items such as plastic instruments, intraocular lenses, and suction catheters. Requires aeration afterwards as it is toxic.
- Formaldehyde gas – Sometimes used for fumigation of operation theatres.
2. Chemical Methods (Disinfection)
Disinfectants are chemical agents used to kill or inhibit microorganisms on non-living surfaces.
- Alcohols (70% ethanol or isopropanol) – Used for disinfecting skin, thermometers, and small instruments.
- Chlorine compounds (sodium hypochlorite) – Used for surface disinfection and cleaning of blood spills in ophthalmic wards.
- Aldehydes (glutaraldehyde) – Used for sterilizing endoscopes, tonometer prisms, and contact lenses when heat sterilization is not possible.
- Quaternary ammonium compounds – Used for cleaning floors and surfaces in hospitals.
- Phenolic compounds – Used for general environmental disinfection.
- Hydrogen peroxide – Used in sterilization systems and also as lens disinfectant in contact lens care.
- Iodophors (povidone iodine) – Used as antiseptic on conjunctiva and periocular skin before ocular surgery to prevent endophthalmitis.
Hospital Application in Ophthalmology
In ophthalmic practice, strict sterilization protocols are vital:
- Operation Theatre – Autoclaved instruments, UV sterilization of air, fumigation with formaldehyde, strict asepsis before intraocular surgery.
- Diagnostic Equipment – Tonometer tips disinfected with alcohol or glutaraldehyde to prevent transmission of adenovirus and herpes.
- Contact Lens Practice – Solutions sterilized by filtration, lenses disinfected using multipurpose solutions or hydrogen peroxide systems.
- Postoperative Care – Eye drops prepared under sterile conditions to prevent microbial contamination.
Laboratory Application
In microbiology labs dealing with ocular samples (conjunctival swabs, corneal scrapings, aqueous humor), sterilization ensures accurate results and prevents cross-contamination.
- Glass slides, test tubes, Petri dishes sterilized by hot air oven.
- Culture media sterilized by autoclaving.
- Biological safety cabinets sterilized with UV light.
- Waste material disinfected with hypochlorite before disposal.
Clinical Importance
Failure of proper sterilization can result in catastrophic ocular infections. For example:
- Postoperative endophthalmitis – Linked to contaminated surgical instruments or intraocular lenses.
- Epidemic keratoconjunctivitis – Spread by inadequately disinfected tonometer tips.
- Pseudomonas keratitis – Associated with poor sterilization of contact lenses and their storage cases.
