PRELIM: BACTERIOLOGY

PROKARYOTES EUKARYOTES ARCHAEOLYTIC

-Before Nucleus -True Nucleus -Melts plastic
-Common in bacteria -Organelles -Ancient times
-Cyanobacteria, For pigment -Human Cells -Gas vesicles
-Cytoplasmic membrane -Animal Cells -Residing at the inner part of the earth
-Plants -S protein, cell wall
-Ex: Methanogen-contains CH3
Halobacterium- Bacteriorhodsin
Thermococcus
Mantoux- skin test for TB
Methylene Blue- Colorless, Gaspak Jar-Anaerobic
Oven- for sterilization
Incubator-set to 31’C or 35-37’C
Refrigerator- 0-4’C
Freezer- -70’C or -20 - -70’C

Microscope- Binocular, Halogen & Tungsten Lamp

Conventional Microscope- with mirror
Parfocal- remain the focus

2 MICROBIAL ISOLATE

Wet mount- Hanging drop

Dry mount- Dead state, Fixed

*If LIVING STATE, do not use the Cedar Oil

*for MEASUREMENT –Micrometer Caliper, 0.2 μm

Chemical Agent
Bacteriostatic
-inhibits/stops the growth of microorganism
Bactericidal
-kills/eliminates microorganisms

AGENTS
Acid or Alkaline solutions
Phenol
Alcohol
Halogen
Salts or Heavy metal

Magnification-product of eyepiece & objective power

-10x or 5x
Scanner- 4x obj. power = 40x OIO- 1000X
LPO- 10x obj. power = 100x *STANDARD= 10X
HPO- 40x obj. power = 400x
HISTORY OF MICROBIOLOGY

RUDOLF VIRCHOW- concept of biogenesis

LOUIS PASTEUR- pasteurization
JOHN TYNDALL- Tyndallization
FERDINAND COHN- discovered bacteria can withstand a series of heating and boiling, ENDOSPORES
ANTOINE LAURENT LAVOISIER- importance of oxygen to life
IGMAZ SEMMELWEIS- routine handwashing
JOSEPH LISTER- system of antiseptic surgery in Britain
ROBERT KOCH- discovered Mycobacterium tuberculosis,1882
-discovered Bacillus athracis,1876
FANNY HESSE- solidifying agent, in the preparation of the culture media.
JULIUS RICHARD PETRI- developed the petri dish
MARTINUS BEIJERINCK & SERGEI WINOGRADSKY- developed enrichment-culture and selective media
EDWARD JENNER- introduced the concept of vaccination
LOUIS PASTEUR- used the term vaccine for an attenuated culture
CHARLES CHAMBERLAND-created a porcelain bacterial filter and anthrax vaccine together with Pasteur
EMIL VON BEHRING- antitoxins for diphtheria and tetanus
ELIE METCHNIKOFF-process of phagocytosis
SELMAN WAKSMAN- discovered streptomycin and neomycin
ALEXANDER FLEMING-discovered the antibiotic penicillin
HOWARD FLOREY- purification of Penicillin
EDWARD ABRAHAM- first to propose the correct biochemical structure of penicillin
PAUL EHRLICH- discovered treatment of syphilis
HANS JANSEN- first compound microscope

BACTERIAL TAXONOMY
CARL VON LINNE- laid down the basic rules for taxonomic categories, Binomial system
DOMAIN- bacteria and archaebacterial
KINGDOM-similar to phyla
PHYLUM- composed of similar classes
CLASS- similar orders
ORDER- similar families
FAMILY- similar genera
GENUS-various of species with common characteristics
SPECIES- basic group or the collection of bacterial strains
SUBSPECIES-species which are subdivided based on phenotypic differences
SEROTYPE- based on serologic differences
BIOTYPE-based on biochemical differences
BIOVARS- characterized by biochemical or physiological differences
EPITHET-proper word for the name of the species
MORPHOVARS-variant prokaryotic strains which differ morphologically
SEROVARS-strains with distinctive antigenic properties
STRAIN- population of organism that is differentiated from population within a particular taxonomic
category
BACTERIAL CELL STRUCTURE

PROPERTIES GRAM-POSITIVE BACTERIA GRAM-NEGATIVE BACTERIA

CELL WALL Thick cell wall is peptidoglycan Thin inner peptidoglycan
SHAPE Spherical, rod-shaped Spherical, oval, straight
METABOLISM chemoorganoheterotrohic Phototrophic,
Chemolithoautotrophic
TEICHOIC ACID Present Absent
ENDOSPORE Present in some groups Absent
PERIPLASMIC SPACE Absent Present
FLAGELLAR STRUCTURE 2 rings 4 rings in the basal body
RESISTANCE TO PHYSICAL High Low
DISRUPTION
RESISTANCE TO LYSOZYME Low High
DISRUPTION
INHIBTION BY BASIC DYES High Low
REPRODUCTION Binary fission Binary Fission

*Gram positive cell wall- very thick protective peptidoglycan (Murein Layer)
- consist of N-acetyl D-glucosamine (NAG), N-acetyl D-muramic acid (NAM)
* Gram negative cell wall- composed of proteins, phospholipids and lipopolysaccharides (LPS)

ACID-FAST CELL WALL- has gram positive cell wall structure

- has Mycolic acid- a strong hydrophobic structure e.g. Mycobacterium
tuberculosis
ABSENCE OF A CELL WALL – do not have cell wall contain sterols in their membrane, but some can have
Mycoplasma and Ureaplasma
PLASMA MEMBRANE- deepest layer of the cell envelope and the internal matrix of the cell.
-site of respiration and photosynthesis

CYTOPLASMIC STRUCTURES
RIBOSOMES- consists of RNA and proteins
-site of protein synthesis
GENOME- appears as a diffused nucleoid or chromatin
PLASMID- double stranded element of DNA associated with virulence
2 kinds of Plasmid- Large plasmid- responsible for the production b-lactamases, for resistance
to b-lactam antibiotics
-Small plasmid-resistant to tetracycline and chloramphenicol
INCLUSION BODIES- serves as energy source of food reserve of the bacteria
ENDOSPORES/ASEXUAL SPORES – aid in survival of bacteria against external conditions
- composed of dipicolinic acid and calcium ions
- examples of endospore formers are Bacillus and Clostridium
TYPES OF SPORES ACCDG TO LOCATION:
TERMINAL SPORE- Clostridium tetani
SUBTERMINAL SPORE- Clostridium botulinum
CENTRAL SPORE- Bacillus anthracis
SPOROGENESIS/SPORULATION- is the process of spore formation
GERMINATION- end of the spore’s dormant stage

CELL APENDAGES
GLYCOCALYX-helps the bacteria to attach to the surface of solid objects or tissue.
-appears as a capsule or a slime layer
CAPSULE- protects the bacteria from the attacks of human system cell since it resists phagocytosis and
desiccation.
SLIME LAYER- made up of polysaccharide polymer
- either inhibit phagocytosis or aid in the adherence of the bacteria to the host tissue
FLAGELLUM- for the survivability and the pathogenic ability of bacteria
- causes bacteria to be motile

ARRANGEMENT OF THE FLAGELLA

ATRICHOUS- without flagellum
MONOTRICHOUS- single flagellum on one end
AMPITRICHOUS- single flagellum on both ends
LOPHOTRICHOUS- tuff/group of flagella on one end or both ends
PETRICHOUS- spread over the whole surface

PILI(FIMBRIA)- hair-like structure that extend from the cell membrane to the external environment
-aid in the attachment of bacteria to surfaces
-virulence factor of attachment is the common or somatic pilus
- essential part of the genetic transfer/conjugation process is the sex pilus.

MOTILE BACTERIA NON-MOTILE BACTERIA

Bacillus subtilis Mycobacterium tuberculosis
Escherichia coli Staphylococci
Helicobacter Neisseria
Campylobacter Klebsiella pneumoniae
Clostridium tetani Bacillus anthracis
Pseudomonas Corynebacterium diptheriae

BACTERIA WITH CAPSULES BACTERIA WITH SPORES

Bacillus anthracis Bacillus
Klebsiella pneumoniae Clostridium
Streptococcus pneumoniae

BACTERIA WITH INCLUSION BODIES OR GRANULES

Corynebacterium Babes-Ernst bodies
Mycobacterium Much’s granules
Nocardia and Actinomycetes Sulfur granules
Pasteurella and Bordetella Bipolar bodies
BACTERIAL GENETICS
DNA- Deoxyribonucleic acid- double stranded helical chain of nucleotides
RNA- Ribonucleic acid- single stranded and short nucleic acid, contains ribose

Replication- duplication of chromosomal DNA for insertion into a daughter cell

- approx. takes 40 minutes for the process of replication

Gene expression- encoding information in genetic elements

Transcription- synthesis of single stranded RNA, converts the DNA base sequence of a gene into an
mRNA molecule.
Translation- genetic code with mRNA molecules is translated into a specific amino acid sequence.
Mutation- change in the original nucleotide sequence of a gene or genes, may be induced chemical or
physical, introduction of a foreign DNA into the cell
Recombination- process which genes are transferred or exchanged between homologous regions,
provides a way to acquire or copy new combinations of biochemical pathways.

MECHANISMS OF GENE TRANSFER

Transformation- involves the recipient cell up taking free DNA, when another bacterial cell dies
Transduction- transfer of bacterial genes by a bacteriophage from one cell to another

Generalized transduction- bacterial DNA incorporated with viral DNA

Specialized transduction-bacterial DNA is adjacent to the viral DNA, packaged into a new virus particle

Conjugation- cell to cell contact

- transfer of genetic material from a donor cell to a recipient cell
- plasmid could be transferred by conjugation

BACTERIAL METABOLISM

Glucose- essential nutrient for energy production

Respiration- efficient ATP generating process in which molecules are oxidized
- Glycolysis (Embden-Meyerhof-Parnas)- first stage in carbohydrates metabolism
- oxidation of glucose to pyruvic acid
- Krebs cycle (Tricarboxylic acid or TCA cycle)- enzyme converts pyruvate into carbon
dioxide
- substrate for this process is the acetyl coenzyme A
Fermentation- does not require O2
- carried out by both obligate and facultative anaerobes
- Alcoholic fermentation- turns sugar to ethanol and C02
- Homolactic fermentation- pyruvate is reduced to lactate, e.g Streptococcus and
Lactobacillus
- Heterolactic fermentation- produces substance lactate
- Mixed acid fermentation- utilizes formic hydrogenlyase
- Butanediol fermentation- pyruvate is converted into acetoin
- Butyric acid fermentation- pyruvate into butyric acid
MICROSCOPY

Phase-contrast Microscope- used to identify medically significant fungi grown in a culture

Fluorescent Microscope- uses fluorochromes (dyes) to stain, utilized to observe of chlamydiae,
legionellae, mycobacteria and fungi
Darkfield Microscope-uses a dark field of condenser that blocks away light, used to detect spirochaetes
(Treponema pallidum)
Electron Microscope- uses electrons instead of light to visualize small objects, useful for studying the
morphology of bacteria
-Transmission electron microscope (TEM) – resolving power is 0.2 nm
- used to examine very thin specimens and
microorganism since it can magnify a specimen a million times
-Scanning electron microscope- scans the surface of the cells or specimens
- revolving power is 200 nm.

BACTERIAL STAINING

Basic Dye- commonly used in bacteriology

- examples are methylene blue, basic fuschin, crystal violet, safranin, and malachite green
Simple Staining- a single stain is used; inoculum size is 10^5 CFU m/L
Differential Staining-divides bacteria into separate groups
- application of primary stains
 - application of mordant
-application of decolorizing agent
- application of the secondary stain/counterstain
Negative Staining- excellent technique for studying bacterial gas vacuoles and viral morphology
-e.g. of staining technique is the use of India Pink or Nigrosin dye.
Gram stain- most commonly used differential stain, utilizes crystal violet as the primary stain while
safranin is the secondary stain or counterstain

REACTION TO GRAM STAINING

PRECAUTIONS DURING GRAM STAINING

- crystal violet dye is rinsed too vigorously prior to the application of iodine, it will not be retained and
will leave the Gram-negative bacteria unstained
- decolorization is prolonged, the Gram-positive complex will be removed and the Gram-positive
bacteria will not be stained.
- decolorization is insufficient, falsely appear as Gram positive cells.
-Safranin dye is left applied for more than 1 minute, Gram positive complex will be eliminated

REASONS WHY GRAM-POSITIVE BACTERIA BECOME GRAM-NEGATIVE BACTERIA

- Removal of MgRNA
- Aged, dying, and autolyzing cells
- Using acidic iodine during staining
- Technical error

ACID-FAST STAIN
- used to stain bacteria that have high lipid contents in their cell walls.
-utilizes carbol-fuschin as the primary stain and methylene blue or malachite green
- Heat is applied as a mordant in the Ziehl-Neelsen method while Tergitol is used in the Kinyoun
method.

DIFFERENTIAL CHARACTERISTICS OF ACID-FAST TO NON-ACID FAST

MODIFIED ACID-FAST STAINING METHOD (MODIFIED KINYOUN)

-useful for the ID of intestinal coccidian oocytes
-ideal for cryptosporidia and cyclospora parasites
-specimen used is stools
-uses acid alcohol (1%H2SO4)

MICROBIAL NUTRITION

AEROBES
-requires oxygen and grow well with room air
- e.g. Bordetella, Brucella, Mycobacteria, Pseudomonas

ANAEROBES
-do not require oxygen to grow
3 TYPES OF ANAEROBES
-OBLIGATE ANAEROBES
- absolutely do not require oxygen because they die
- e.g. Clostridium, Bacteroides
FACULTATIVE ANAEROBES
-most clinically significant bacteria
-grow either in the presence or absence of oxygen
-e.g. Enterobacteriaceae
AEROTOLERANT ANAEROBES
-can survive in the presence of oxygen but will be unable to perform metabolic processes
-e.g. Propionibacterium acnes
MICROAEROPHILE
-requires 2% to 10% oxygen for growth
-e.g. Campylobacter, Treponema pallidum