Overview of Microbiology

Overview of Microbiology

• Microbiology studies microscopic organisms too small to see with the naked eye.
• These microorganisms include a large and diverse group, existing as single cells or in clusters.
• Objects smaller than 1 mm typically require a microscope for examination.
• The term microbiology originates from Greek roots: micros (small), bios (life), and logos (science), signifying the study of microscopic life.
• Microorganisms are diverse and ubiquitous, appearing almost everywhere.
• Medical microbiology specifically focuses on pathogenic microbes and their roles in human illnesses.
• Some microbes studied in medical microbiology are Bacteriology (prokaryotes, single-celled), Virology (non-cellular, parasitic infectious agents), Mycology (fungi like molds, yeasts, and macroscopic mushrooms). Protozoology focuses on single-celled organisms.
• Microbes are helpful for recycling elements, fixing nitrogen, photosynthesis, water recycling (sewage treatment), bioremediation , and producing food and drinks.
• Food spoilage, bioterrorism, and disease are harmful impacts from microorganisms.
• Pathogens are microorganisms that can potentially cause disease.
• Infection involves the invasion and multiplication of pathogens in an individual or a population.
• Disease occurs when an infection damages an individual's vital functions or systems, and infection does not always result in disease.

History of Microbiology

• Babylonians used yeast to brew beer.
• Acetic acid bacteria were used for vinegar production over 6000 years ago.
• Romans hypothesized around 2000 years ago that tiny animals caused diseases.
• Real progress began in the 1600s with the creation of basic lenses and microscopes.

Antony van Leeuwenhoek (1632-1723)

• Leeuwenhoek was a founding figure in microbiology, microscopy, and zoology, despite having no formal scientific training.
• He crafted his own lenses and microscopes to examine objects.
• In 1677, Leewenhoek observed microorganisms from canal water under his microscope, which he called "animalcules". Today they are known as protists.

Spontaneous Generation vs. Biogenesis

• Spontaneous generation is the idea that living things could arise from nonliving matter, and was supported by Aristotle and other philosophers since 300 BC.
• One example of an observation that supported this was decaying meat created maggots and flies or that grain created frogs.
• Biogenesis states that living cells can only come from existing living matter.

Francesco Redi (1626-1697)

• Redi (biogenesis) conducted experiments with decaying meat in jars to challenge spontaneous generation.
• Maggots only appeared in open jars, leading to the conclusion that maggots were offspring of flies, not spontaneous products.

John Needham (1731-1781)

• Needham claimed the presence of a "life force" causing spontaneous generation.
• Soup was boiled briefly and poured into clean, cork-lidded flasks, resulting in microbial growth.
• It was concluded that microbes spontaneously arose in broth from a life force.

Lazzaro Spallanzani (1729-1799)

• Spallanzani (biogenesis) argued that Needham's organisms came from heat-resistant microbes.
• No growth occurred when flasks were boiled for a longer duration (1-2 hours).
• The conclusion was that microbes were introduced from the air.

Louis Pasteur (1822-1895)

• Pasteur (biogenesis) believed life originated from previous life and demonstrated microorganisms in the air.
• Skeptics argued overheating killed the "life force".
• Pasteur used swan-necked flasks, which allowed air to enter but prevented bacterial and fungal spores from entering.
• The experiment consisted of boiling broth in flasks to sterilize it and letting it cool, the broth remained free of contamination. If the neck was broken off and then boiled the broth became contaminated.
• Pasteur developed pasteurization which is a process of heating juice at 62.8°C for half an hour.
• Pasteur isolated the anthrax causing bacilli.
• Pasteur developed the rabies vaccine.

Other Microbiology Figures

• John Tyndall (1820-1893) demonstrated that dust carries germs and absence of dust causes sterility.
• Martinus Willium Beijerinck (1851 – 1931) developed the enrichment technique.
• Walther Hesse and Fannie E. Hesse (1883) used agar instead of gelatin in media.
• Joseph Lister (1878) developed pure culture technique.
• In 1928 Alexander Fleming developed penicillin from mold.

Germ Theory of Disease

• Diseases result from the invasion of the body by microorganisms.
• Robert Koch established "scientific principles" in 1876 to demonstrate a cause and effect relationship between a microbe and a disease.
• These principles are known as Koch's postulates.
  • The same microbe is always associated with a specific disease.
  • The microbe must be isolated from the host with the disease and grown in a pure culture.
  • The specific disease must be reproduced when a pure culture is inoculated into a healthy host.
  • The original microbe must be recovered from the experimentally infected host.
• Koch determined the etiology of cholera, tuberculosis and anthrax.

Exceptions to Koch's Postulates

• It is sometimes impossible to grow organisms on artificial media. For example, you cannot grow Treponema pallidum (Syphilis) or Mycobacterium leprae (Leprosy).
• All viruses cannot be grown on artificial media.
• There are ethical concerns about infecting subjects with infectious agents.
• Some diseases have no animal models.

Normal Flora (Microbiota)

• Normal flora are a mixture of microorganisms regularly found at anatomical sites in a healthy person.
• Some microorganisms are found in human hosts only.
• Normal flora can be found in the environment.
• Factors influencing the normal flora include genetics, age, sex, stress, nutrition, and diet.
• Resident flora are quick to reestablish itself if disturbed, colonization from other microorganisms is possible if they are disturbed, and this kind of flora is more important than other flora.
• Transcient flora are non-pathogenic or potentially pathogenic microorganisms on skin or mucus membranes for days or weeks, derive from the environment, don't cause disease, don't establish itself quickly on the surface and are of little significance.
• Examples of where normal flora can be found are the nose, mouth, throat, large intestine, vagina, urethra, and skin.
• Normal flora synthesizes and excretes key vitamins, prevents colonization by pathogens by competing for essential sites, and produces bacteriocins to kill other bacteria.
• If the normal flora is disrupted, this leads to an imbalance in the bodies bacteria which can lead to yeast infections and diarrhea.
• Broad spectrum antibitics can lead to a disruption of normal flora.

Normal Flora of the Skin

• Low pH, fatty acids, and lysozyme help in eliminating non-resident skin microorganisms.
• Neither extensive sweating, bathing, or washing can eliminate or significantly change the normal resident skin flora.
• Staphylococcus epidermidis is the major inhabitant, making up over 90% of the skin flora.
• Staphylococcus aureus can be found in the nose, perineum, and vulvar skin. The occurrence varies with age.
• Benefits of Normal Flora are the production of antimicrobial peptides, inhibition of fungal growth, wound healing, inflammation control, and immune system development. Harmful aspects are body odor, acne, and opportunistic infections.

Normal Flora of the Respiratory System

• Lower respiratory tract is usually sterile.
• Normal flora is present in the nose, sinuses, oropharynx, and pharynx.
• Airways are lined with ciliated epithelium that remove debris through the mucus blanket.
• Normal flora make bacteriocins that harm pathogens and keep the body ready for infection.

Normal Flora of the Mouth

• At birth the mouth is sterile.
• Within 4-12 hours of birth, lactobacilli and streptococci grow.
• Teeth allow for Streptococcus mutans and Streptococcus parasanguis.
• Gingival crevices may contain anaerobic species and yeasts.
• Puberty allows for growth of Bacteroides and spirochetes.
• Saliva is 108 bacteria/mL with >700 species.
• Oral flora is useful in competing with pathogens and stimulating immunity.
• Harmful flora cause plaque and dental disease.

Normal Flora of the Gastrointestinal Tract

• Colon contains > 1011 bacteria mL-1, Stomach contains 102 -104 bacteria mL-1, Small intestine contains 107 - 108 bacteria mL-1.

Normal Flora of the Genitourinary Tract

• Upper urinary tract (kidneys, ureters, bladder) is usually sterile.
• Male anterior urethra contains enteric and enterococcus.
• Vaginal microbiota varies with life stage: at birth it's like mother's flora, in neonates skin + enteric + strep (pH 7), at puberty Lactobacilli+same as skin+anaerobes+strep (pH 5), after menopause return to prepuberty.
• Normal flora of the genitourinary tract competes with pathogens and produces bacteria that inhibits pathogenic yeast.

Definition of Terms

• Sterilization is the complete removal of microorganisms, including spores.
• Disinfection is the process of making a substance or object non-infectious.
• Disinfectants are products or biocides used to kill microorganisms on surfaces/objects.
• Septic is the presence of pathogenic microbes in living tissue.
• Antiseptic is a substance that destroys or inhibits the growth of microorganisms in or on living tissue.
• Aseptic is characterized by the absence of pathogenic microbes.
• Preservation is the prevention of multiplication of microorganisms in pharmaceuticals or foods.
• Germicide kills vegetative forms of microorganisms but does not necessarily kill the spores.
• Sanitizer kills 99.9% of microorganisms on objects.

Sterilization Factors

• Organisms die differently amongst eachother.
• Killing one is easier than many.
• Organic material may prevent sterilization.

Disinfection Chemical Types

• Heat is 75°C to 100°C for 30 mins
• Glutaraldehyde is 2%-3.5%
• Hydrogen peroxide is 3%-25%
• Formaldehyde is 3%-8%

Methods of Sterilization

• There are two sterilization methods (physical and chemical).
• Physical agents sterilize through dry and moist heat, radiation, or filtration.
• Chemical agents sterilize/disinfect through animate and inanimate products.

Physical Methods of Sterilization - Heat Sterilization

• Heat sterilization is very effective and can be dry (cheaper) or wet/moist (more effective).
• Degradation of organic matter is the main effect that heat provokes, especially proteins.

Physical Methods of Sterilization - Moist Heat

• Moist heat is more effective than dry heat; denaturation of proteins happens at lower temperatures.
• Types of moist heat include boiling and autoclaving.
• Steam under pressure is pure and dependable.
• This is done at 121°C and 15 pounds of pressure, for 15 minutes
• Sterilizing at these conditions is effective, except for heat-labile substances.
• Autoclaving requires adequate contact, high temperatures, and time.
• Autoclaving requires a continuous source of heat/sterilizer, there needs to be adherence to strict time/temperature settings, repeated sterilization can dull tools, and it cannot withstand high temperatures.
• Intermittent boiling (Tyndallization) sterilizes for 30 minutes at 100 °C over three days and sterilizes substances that would be damaged by autoclaving. This process kills vegetative cells with spores and uses heat to induce spores.
• Pasteurization is effective in reducing microbial contamination using mild heat. There is the batch method (62.8° for 30 minutes) and flash point (71.7 for 15 seconds then cooled quickly).

Physical Methods of Sterilization - Dry Heat

• If there is high heat or hot air, this will kill microorganisms effectively.
• The oxidation of organic material is the main effect of dry heating, and it requires higher temperatures/longer times.
• Red hot heat is done to loops and straight-wires in microbiology labs to completely oxidize infectious agents on metal/glass surfaces.
• Flaming is the passing of materials through a flame.
• Incineration burns any organism into ash and is used to sanitize biohazardous waste.
• Ovens work at higher temperatures with prolonged heating, and it is used to sterilize glass and metal.
• In dry heat applications, there is limited versatility and low speed versus moist heat. This also dries out material over time.

Physical Methods of Sterilization - Radiation

• Ionizing is related to the wavelength. Shorter is more energy.
• Gamma rays and UV light are examples of high-energy radiation.
• There are two forms of radiation - ionizing and non-ionizing.

Physical Methods of Sterilization - Ionizing Radiation

• This method includes high beams/gamma rays/x-rays to cause molecule ionization, producing highly destructive hydroxyl radicals to organic cellular compounds.This is able to penetrate packaging to sterilize contents.
• Water is split into OH- + H+ and electron (Hydroxyl radicals and Hydrogen ions respectively).

Physical Methods of Sterilization - Non-ionizing Radiation

• Longer UV lights exist with x and gamma rays.
• UV light excites non-irradiated molecules more so than others.
• Wavelengths at about 260 nm achieve greatest bactericidal properties via nucleotides.
• Two adjacent pyrimidines on DNA are bonded, it is used in air sterilization, and UV light has very little penetration in objects.
• This sterilization method is commonly used for air purification, surfaces, operating rooms, and sterile product rooms.

Physical Methods of Sterilization - Filtration

• This method involves sterilizing liquids by removing microorganisms that can’t be sterilized using heat.
• Filters use ceramic and cellulose.

Chemical Sterilization - Antiseptics

• These agents (mercurials, silver nitrate, iodine solution, alcohols, detergents) are used on the skin and mucous membranes to remove or kill microorganisms.
• They should be used on surgical scrubs or skin and cervical preps before a clinic process.

Chemical Sterilization - Disinfectants

• These agents are used on instruments and surfaces to kill microorganisms (chlorine/glutaraldehyde)
• They are indicated for processing of instruments and surfaces and should not be used on skin.

Disinfection - Phenol

• Is known as carbolic acid, and is one of the first antiseptics.
• This alters selective permeability through the inactivation of cytoplasmic enzymes
• As disinfectants is known to be toxic with unpleasant odors.

Disinfection - Alcohol

• Alcohol works through protein denaturation, and destroys lipid/cell membranes and mechanical removal.
• Common use is with isopropyl alcohol, and using surgical instruments (not sporicidal).

Disinfection - Aldehydes

• These have a broad spectrum of activity, and includes formaldehyde and glutaraldehyde, sterilizing to high-level disinfectants.
• They are often used as a fixative in electron microscopy, or for surgical equipment, and are very toxic/carcinogenic.

Disinfection - Halogens

• These (Iodine/Chlorine) are strong oxidizing agents working to destroy cells, and is found in many of the other chemicals.
• Iodine destroys metabolic compounds and is rapidly bactericidal/virucidal
• Chlorine is Bactericidal, sporicidal, virucidal used in water supplies, and swimming pools due Oxygen radicals, from sodium hypochlorite.

Disinfection - Heavy Metals/Their Compounds

• Compounds include mercury, lead, zink, silver, and copper and inactivate enzymes/proteins by combining with them.
• Silver is found in 1% silver nitrate. These disinfect the eyes.
• Zink is fungicidal for athlete's foot, copper sulfate to kill water algae, and mercury has a great affect on treating minor cuts.

Disinfection - Oxidizing Agents

• Hydrogen-peroxide is used for sterilization, in high amounts for longer than most with viruses.