Scope and Significance of Microbiology

Scope of Microbiology

• Bacteriology involves the study of bacteria.
• Mycology involves the study of fungi.
• Virology involves the study of viruses.
• Parasitology involves the study of parasites.

Importance of Microbiology

• Microorganisms are found everywhere.
• Normal flora/indigenous flora consists of thousands of organisms inhibiting the human body
• Some microbes are essential in biotechnology.
• Some bacteria and fungi are important sources of antimicrobial agents.
• Some microbes function as saprophytes or decomposers.
• The study of microbes has provided insights into disease production, improving disease management and control.

Significance of Microbiology

• Medical Microbiology focuses on microbes, their infections in humans, and related aspects like disease, diagnosis, prevention, and treatment.
• Study of microbiology helps the principles of hospital and community hygiene maintenance and prevention.
• It contributes to reducing mortality and morbidity and the duration of hospital stay for inpatients.

Evolution of Microbiology

• Archeologists and evolutionists discovered evidence of primitive microorganisms from 3.5 Billion years ago.

Robert Hooke (1635 – 1703)

• Robert Hooke discovered the cell.
• Robert Hooke heralded the cell theory, that living organisms are made up of cells.

Anton Van Leeuwenhoek

• Anton Van Leeuwenhoek, the father of microbiology, developed a single-lens microscope.
• He used this microscope to observe microbes, which he called animalcules.

Louis Pasteur (1822 – 1895)

• Louis Pasteur introduced the germ theory of disease.
• Microbes can cause infectious diseases.
• Louis Pasteur developed the pasteurization process.
• Louis Pasteur introduced the terms aerobes and anaerobes.
• Louis Pasteur developed the fermentation.

Robert Koch (1843 – 1910)

• Robert Koch proved that microorganisms transmit disease.
• A pathogen must be present in every disease case.

Edward Jenner

• Edward Jenner responsible for the smallpox vaccine.

Joseph Lister

• Joseph Lister responsible for the development of antiseptic surgery.
• Antibiotics were introduced to the medical world after World War 2.

Alexander Fleming

• Alexander Fleming discovered penicillin.
• Penicillin came from the Penicillium notatum/rubens mold

Microscopes

• Microscopes serve as optical instruments that allow visualization of the microbial world.
• Lenses in microscopes produce magnified images which allow visualization of the form and structure of these tiniest of organisms.

Slides

• Microscope slides are used to hold specimens for viewing under compound light microscopes.
• The slides include dry mount, wet mount, prepared slides, and smears.
• Different types of slides include dry mount, wet mount, prepared slides and smears.

Autoclave

• An autoclave is the most effective method of sterilization.
• Bacillus Stearothermophilus at 121°C, 15 PSI for 15 minutes is a biological indicator of autoclave effectiveness.

Differential Stain

• Differential stains are used to distinguish one group of bacteria from another.

Gram Stain

• Gram stain distinguishes gram-positive bacteria from gram-negative bacteria.
• Gram-positive bacteria stain blue or purple.
• Gram-negative bacteria stain red or pink.

Gram Stain General Rule

• All cocci bacteria are gram-positive except Neisseria, Branhamella, Moraxella and Veillonela.
• All bacilli bacteria are Gram (-) except Bacillus, Bifidobacterium, Mycobacteria, Actinomyces, Clostridium, Corynebacterium, Propionibacterium, Erysipelothrix, Listeria, Lactobacillus. Nocardia

Gram Stain Reagents and Colors

• Primary stain (crystal violet) stains Gram-positive bacteria purple.
• Gram stain mordant (Gram's Iodine) stains Gram-positive bacteria purple.
• Decolorizer (Acetone/ Alcohol) stains Gram-positive bacteria purple and Gram-negative bacteria colorless
• Secondary stain (Safranin) stains Gram-positive bacteria purple and Gram-negative bacteria red or pink

Acid-Fast Stain

• Acid-fast stains used for bacteria with high lipid content in their cell wall, which cannot be stained using gram stain.

Ziehl-Neelsen stain

• Ziehl-Neelsen stain, also known as the "hot method", uses steam-bathing of the prepared smear after adding the primary dye.
• Acid-fast organisms will appear red on a blue background.

Kinyoun Stain

• Kinyoun stain, also known as the "cold method", does not use heat after adding the primary stain.
• Acid-fast organisms will appear red on a green background when Kinyoun stain is used.

Culture Media

• Media are used to grow microorganisms.
• Culture medium refers to an aqueous solution that contains all necessary nutrients essential for the growth of microorganisms.

Culture Medium Primary Levels of Categorization

• Physical state
• Chemical composition
• Functional type

General Purpose Media

• General purpose media supports primary isolation of a broad spectrum of microbes.
• It contains a mixture of nutrients that support the growth of both pathogenic and non-pathogenic microbes.
• Examples of general purpose media: peptone water, nutrient broth, and nutrient agar.

Enrichment Media

• Enrichment media contains complex organic substances such as blood, serum, or special growth factors.
• Increase the number of desired microbes without stimulating the rest of the bacterial population.
• Used to grow fastidious or nutritionally extracting bacteria.

Blood Agar

• Blood agar has general nutrients with 5% - 10% (volume) blood added to a blood agar base.
• Gram-positive bacteria produce exotoxins in blood agar that cause hemolysis of RBC.

Hemolytic Reactions

• Beta hemolysis leads to complete lysis of RBC, resulting in complete clearing around colonies.
• Alpha hemolysis leads to incomplete lysis of RBC, producing greenish discoloration of the blood agar.
• Gamma hemolysis means no hemolysis and no change in the medium.

Selective Media

• Selective media has one or more substances that encourage the growth of a specific target of microbes and inhibit the growth of others.
• They are usually agar-based solid media that allow isolation of individual bacterial colonies.

Mannitol Salt Agar

• Mannitol Salt Agar contains 10% NaCl and is used for the isolation of Staphylococcus aureus.

MacConkey's Agar

• MacConkey's agar promotes the growth of gram-negative bacteria.
• MacConkey's agar inhibits the growth of gram-positive bacteria.

Transport Media

• Transport media is used for clinical specimens needing immediate transport to the lab after collection.
• Transport media prevents drying of the specimen and inhibits the overgrowth of commensals and contaminating organisms.
• Cary Blair transport medium is one type of transport medium.

Virology Key Topics

Bacteria vs. Viruses

• Bacteria replicate through binary fission/mitosis, viruses use the host's nucleus.
• Bacteria contain both DNA and RNA, viruses also contain DNA/RNA
• Bacteria contain mitochondria as Eukaryotes or are absent as Prokaryotes, whereas viruses are absent

Viruses General Characteristics

• Viruses are obligate intracellular parasites and cannot self-replicate.
• Viruses induce the host cell to synthesize virus particles once inside living cells.
• The genome is either DNA or RNA (single or double stranded).
• Viruses cannot produce ATP.
• Viruses range in size from 25 to 270 nm.
• Classifying viruses is based on nucleic acid type, virion size and shape, and presence or absence of an envelope.

Viral Structure

• The virion is the entire viral particle.
• The capsid is the protein coat that encloses the genetic material.
• The capsomer is the protein subunit of the capsid.
• The nucleocapsid includes of the capsid and genetic material.
• The envelope is the outer coating and naked nucleocapsids have no envelopes.

Viral Replication

• Adsorption involves attachment of the virus to a specific receptor on the host cell.
• Penetration involves the entry of the virus into the host cell.
• Uncoating involves separation of the capsid from the genome or rearrangement of the capsid proteins, which exposes the genome for transcription and replication.
• The eclipse period is the stage when the genetic material is replicated, but intact virions are not detectable.
  • mRNA codes for viral proteins and enzymes necessary for nucleic acid synthesis during the eclipse period.
• Assembly (maturation) involves genetic material assembled into a protein coat.
• Viruses are released from the host cell.
  • Cell lysis: Naked viruses lyse host cells and exit through a hole in the plasma membrane.
  • Budding: An intact virion pushes outward from a host's membrane.
  • The membrane around the virion is cleaved and resealed, thus becoming the viral envelope.

Diagnosing Viral Diseases

Specimen Processing Considerations

• Viruses are in the highest concentrations during the first several days following symptom onset.
• Samples are typically collected early in the disease course and should generally come from the infected site.
  • Skin infections: Collect from the rash site, serum, or urine, depending on the virus.
  • Respiratory infections: Obtain sputum or throat swabs.
  • Central nervous system infections (e.g., meningitis and encephalitis): Collect cerebrospinal fluid (CSF) and serum for meningitis, along with stool or throat swabs. Brain biopsy material and sometimes serum for encephalitis.
  • Urogenital infections: Use needle aspirates and endocervical and urethral swabs.
  • Gastrointestinal tract infections: Obtain stool samples and rectal swabs.
  • Eye infections: Use eye swabs and corneal scrapings.

Sample Transport

• Viral culture samples should be placed into a viral transport medium (VTM).
• VTM contains buffered saline, protein stabilizers, and antimicrobials that inhibit bacterial and fungal growth.
• Viral culture samples can be refrigerated in VTM for about 48 hours.
• Samples should never be frozen at -20°C, but can be stored at -70°C, although infectivity will be diminished.

Key Medically Important DNA Viruses:

• Poxviridae: Variola virus, molluscum contagiosum virus
• Herpesviridae: Herpes simplex viruses types 1 and 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesviruses 6, 7, and 8
• Adenoviridae: Adenovirus
• Hepadnaviridae: Hepatitis B virus
• Papillomaviridae: Papillomavirus
• Polyomaviridae: JC and BK viruses
• Parvoviridae: Parvovirus B19

Major Clinical Syndromes of Human Herpesviruses and Latent Infection Sites

Herpes simplex virus Type 1:

• Gingivostomatitis in children and young adults, recurrent oral-labial infection (cold sores), infection of the cornea (keratitis), herpes encephalitis are major clinical syndromes.
• Site of Latent Infection is the trigeminal nerve root ganglion and autonomic ganglia of superior cervical and vagus nerves.

Herpes simplex virus Type 2:

• Genital herpes, neonatal herpes are major clinical syndromes.
• Site of Latent Infection Sacral nerve is the root ganglia.

Varicella-zoster:

• Chickenpox (primary infection), shingles or zoster (reactivation) are major clinical syndromes.
• Site of Latent Infection is the thoracic, cervical or lumbar nerve root ganglia.

Cytomegalovirus:

• Asymptomatic infection, heterophile-negative mononucleosis, fever hepatitis syndrome in neonates and transplant patients, interstitial pneumonia in immunocompromised patients are major clinical syndromes.
• Site of Latent Infection is Leukocytes (neutrophils and lymphocytes).

Epstein-Barr virus

• Heterophile-positive mononucleosis is a major clinical syndrome.
• Site of Latent Infection is the B lymphocytes.

Human herpesvirus 6:

• Roseola (sixth disease) is a major clinical syndrome.
• Site of Latent Infection is Peripheral blood mononuclear cells.

Human herpesvirus 7:

• Roseola and febrile diseases in children are major clinical syndromes.
• Site of Latent Infections are Peripheral blood mononuclear cells.

Human herpesvirus 8:

• Kaposi's sarcoma is a major clinical syndrome.
• Site of Latent Infection is Peripheral blood mononuclear cells.

Key Medically Important RNA Viruses:

• Paramyxoviridae: Measles, mumps, respiratory syncytial, parainfluenza, and metapneumo viruses
• Orthomyxoviridae: Influenza A, B, and C viruses.
• Coronaviridae: Coronavirus
• Arenaviridae: Lymphocytic choriomeningitis and Lassa fever viruses.
• Rhabdoviridae: Rabies virus
• Filoviridae: Marburg and Ebola viruses
• Bunyaviridae: California encephalitis, Hantaan, sin nombre, and Crimean-Congo viruses.
• Retroviridae: Human T lymphotropic and human immunodeficiency viruses.
• Reoviridae: Rotavirus and reovirus
• Picornaviridae: Rhinovirus, poliovirus, enterovirus, ECHO virus, coxsackievirus, hepatitis A virus
• Togaviridae: Rubella virus and western, eastern, and Venezuelan equine encephalitis viruses.
• Flaviviridae: Yellow fever, dengue, St. Louis encephalitis, hepatitis C, and West Nile viruses.
• Caliciviridae: Norwalk and Sapporo viruses.

Important Human Hepatitis Viruses

• Hepatitis A genome is RNA, is transmitted via Fecal-oral, has no chronic liver disease and is rarely fatal, but severity increases with age.
• Hepatitis B genome is DNA, is transmitted via Parenteral, blood, sexually, needles, perinatal, and has 5-10% chronic hepatitis associated with hepatocellular cancer.
• Hepatitis C genome is RNA, has Parenteral, blood, needles, perinatal transmission and chronic infections are common.
• Hepatitis D genome is RNA, has Parenteral, blood, sexually, needles, perinatal transmission, and coinfection/superinfection in patients infected with HBV.
• Hepatitis E genome is RNA, is transmitted via Fecal-oral, and has a wide range of clinical outcomes but has a high mortality rate among pregnant women.

Definition of Fungi

• Fungi include yeast, mushrooms, and molds.

Methods of Fungi to Survive

• Saprobes live on decaying matter.
• Symbionts have Mutually advantageous relationships.
• Commensals live in close proximity, where one organism benefits, and the other is unaffected.
• Parasites live in or on a host causing harm.

Fungi Characteristics

• Fungi have a cell wall composed of chitin.
• Fungi are eukaryotes with a true nucleus.
• Fungi are masters of recyclers.

Mycology Terms

• Moulds: Multicellular fungi
• Yeasts: Single-cell fungi
• Mycosis: Fungal infection
• Systemic mycosis: Multiorgan infection caused by fungi
• Opportunistic mycosis: Fungal disease that is seen primarily in immunocompromised patients
• Dimorphic fungi: Fungi that show both a non-mould (e.g., yeast) and mold phase

Fungal Structure

• Hyphae are long, branching filaments that come together to form mycelium.
  • Septate hyphae have cellular separation or cross-walls.
  • Sparsely septate (formerly aseptate) hyphae contain few or any cellular separations.
  • Coenocytic refers to hyphae lacking cross-walls.
• Pseudohyphae are a chain of cells formed by budding that resemble true hyphae.
  • Pseudohyphae are constricted at the septa, form branches with septation, and have terminal cells smaller than other cells.

Reproduction

• Sexual reproduction requires the formation of specialized fungal structures called spores.
  • Fungi undergoing sexual reproduction are termed perfect fungi.

Two Types of Spores

• Ascospores: are spores contained in a saclike structure.
• Basidiospores: are Spores contained in a club-shaped structure.

Sexual Reproduction:

• Asexual reproduction involves division of the nucleus and cytoplasm only.
• Fungi that undergo asexual reproduction are termed imperfect fungi and is the only fungal group to produce conidia.
• Oospores are spores resulting from the fusion of cells from two different hyphae.
• Zygospores are spores resulting from the fusion of two identical hyphae

Yeast Introduction

• Yeasts commonly cause vaginitis and urinary tract infections (UTIs) in women and can cause diseases in healthy and immunosuppressed individuals.
• Yeast can cause newborn infections and meningitis.
• Candida albicans is most common cause of yeast infections.

Parasitology

• Commonly isolated parasites worldwide are:
  • Enterobius vermicularis
  • Ascaris Lumbricoides
  • Trichuris trichuria

The Triad of Infection/Unholy Tree

• Hookworm
  • Ascaris Lumbricoides
  • Trichuris trichuria

Parasites with Heart to Lung Migration and/or are Soil-Transmitted Helminths

• Ascaris Lumbricoides
• Strongyloides
• Hookworm
• Trichuris trichuria

Ascaris Lumbricoides

• The giant intestinal roundworm
• Mode of Transmission is ingestion of fertilized embryonated eggs
• Diagnostic stage is when the egg.
• Can lead to Loefflers disease, highly eosinophilic, worm bolus and pulmonary obstruction.

Enterobius Vermicularis characteristics

• Referred to as pinworm, sitworm, society worm.
• It is D-shaped, has the MOT of Inhalation and ingestion of embryonated eggs.
• Diagnostic stage is egg
• Pathological infection include Pruritis Ani

Tricuris Tricuria characteristics

• Causes whipworm from ingesting embryonated egg.
• The diagnostic stage is the egg.

Capillaria Philippinensis characteristics

• Causes Pudoc worm
• Can be transmitted by Ingestion and was first discovered in Ilocos Norte.
• The diagnostic stage egg.
• The Intermediate host: (Ipon) most common Bagsang, Bagtu, Birot

Hookworm Characteristics

• Necatur Americanus (new world hookworm)
• Ancylostoma duodenale (old world hookworm)
• Transmitted via Skin penetration
• Infective stage and Diagnostic stage: egg

Strongyloides Stercoralis characteristics

• Threadworm has Skin penetration
• The infective stage is Filariform larva/ L3