MICROBIOLOGY Reference: Clinical Laboratory Science by Linne and Ringsrud, published by

Elsevier Chapter 15 (7th edition)/ page 475-538. (Student Procedure Worksheet 15.1-15.4) .

Objectives

Following this course, the student will be able to:

1. Know and recognize the various microbiological procedures.

2. Know the basics of staining procedures.

3. Know the method of manufacture of stains and reagents (including media).

4. Know the various types of cultural methods and media (aerobes, anaerobes, etc.).

5. Make smears for identification purposes.

6. Perform basic staining techniques such as Gram stain, ZN, etc.

7. Collect blood specimens for blood culture and know the techniques involved.

8. Know the various types of containers used for specimens.

9. Know the different types of swabs encountered in microbiology - e.g., throat,

nasopharyngeal.

10. Understand routine testing for anaerobic organisms and methods used to obtain an
anaerobic environment (Gas Paks, glove box, candle jar and incubator).

11. Know the methods of sensitivity testing (Kirby-Bauer, M.I.C.).

12. Know the basics of collection and plating out of mycology samples.

13. Know the basics of virus structure and list the steps required to infect the host.

14. Know the techniques involved in concentration and staining methods for fecal parasites.

Microbiology is the study of microorganisms including algae, bacteria, rickettsia, fungi, viruses
and protozoa. So you can see that even in such a definition, there are divisions in the field of
microbiology. The history of microbiology dates from about 1683 when Anton Van
Leeuwenhoek, he had experimented with lenses, making it possible to see the various shapes of
bacteria. This led to the Microscope development. Van Leeuwenhoek was possibly the first
person to make a microscope. He has been named the "Father of Microbiology". Microbiology,
as a science, progressed through the work of Louis Pasteur, who performed a number of
experiments using various nutrients to foster the growth of Microorganisms. Since that time,
there have been a great many discoveries, in the field of Microbiology and antibiotic industry to
eliminate microbial infections.

Another great scientist is, Robert Koch who gave us a great amount of knowledge with regard
to disease causing organisms (pathogens). He isolated and identified many organisms causing
infectious diseases (for many years Mycobacterium tuberculosis was called Koch's bacillus).
There are now many other methods that can be used in Microbiology. General characteristics
about the Microorganisms:

1. The microorganism must be found in all cases of the disease. 2. The microorganism must be
isolated from the animal and grown in a pure culture in the laboratory. 3. The microorganisms
from the pure culture, when injected into a healthy, susceptible animal, will produce the same
disease as in the original infected animal. 4. The microorganism, isolated and cultured from the
injected animal, must be the same as the one in the original infected animal.

Microorganisms are considered to be transitional forms of either plant or animal origin, and are
placed in a kingdom called the Protista. Within the Protista, there are two cell classes,

1) The higher protista (algae, Protozoa, fungi and slime molds) and

2) the lower protista (bacteria, blue green algae, rickettsia and viruses). We are interested in
the bacteria. Because bacteria are so small, they require special techniques such as staining or
electron microscopy to reveal their sizes, shapes and cellular structure.

Many bacteria are beneficial to man (aquatic bacteria, agricultural bacteria, environmental
bacteria, space bacteria); the medically important bacteria are those that cause disease
(pathogenic). Bacteria are single-cell organisms that are spherical; rod shaped (straight or
curved) or spiral in form. Motile by means of flagella and non-motile forms exist. The average
size range is 0.2 -5.1 microns. Organisms may be free living, saprophytic, parasitic or
pathogenic.

The type of bacterium that exists on all types of materials is known as free living.

 A saprophyte is an organism that can exist on dead organic (heterotrophic) or inorganic

(autotrophic) matter.

a. Heterotroph is defined as an organism that requires carbon in organic compounds found in

plants and animals. (Heterotroph literally means feeding on others).

b. Autotroph is defined as a self-nourishing. Microorganisms in this category, do not require

organic carbon and nitrogen as a source of energy, but are able to form carbohydrates and
protein from carbon dioxide and inorganic salts.
Parasitic, the organism has a symbiotic existence, i.e., is totally dependent on another organism
or animal tissue.

 Pathogenic, is defined as disease causing.

Bacterial growth starts with the inoculation into suitable media (correct environment). The
growth is in numbers rather than in size, as organisms reproduce quickly through the binary
fission. Video: http://www.britannica.com/science/binary -fission

There are four main phases of growth.

1. Lag phase. Organism adaptation to its environment. This is a slow period of growth and there
is variation in the time involved according to the organism involved.

2. Log or exponential phase. Organisms divide at a constant rate. Growth rate is linear. As you
can imagine, growth rate is logarithmic.

3. Stationary phase. Because of the waste produced (Toxic) and the use of nutrient available
bacteria, stop the log phase and form a plateau. The number of organisms stays steady at this
stage.

4. Phase of decline or death phase. Bacterial Cells at this stage begin to die. The causes of death
are the same as in the stationary phase, but the total number of organisms may remain the
same.
Cell Structures

STRUCTURE DESCRIPTION FUNCTION Cell Membrane or Plasma membrane Lipid bilayer

throughout which a variety of proteins are distributed in a mosaic pattern. Protection; regulates
passage of materials into and out of cell; helps maintain cell shape; communicates with other
cells intracellular fluid (Cytoplasm aka) Mitochondria Sacs consisting of two membranes; inner
membrane is folded to form cristae

Site of most of the reactions of cellular respiration; power plants of cell

Ribosomes Non membranous granules composed of RNA and protein; some attached to ER

Manufacture protein

Endoplasmic Reticulum Rough

Network of internal membranes extending through cytoplasm; forms system of tubes and
vesicles In rough ER, ribosome stud outer surfaces

Intercellular transport of materials; manufactures and transports proteins

Endoplasmic Reticulum Smooth

Network of internal membranes extending through cytoplasm; forms system of tubes and
vesicles Smooth ER lacks ribosome on outer surfaces

Intercellular transport of materials; produces steroids in certain cells; conduction of impulses in

muscle cells

Golgi Apparatus (Complex)

Lysosomes

Stacks of flattened membranous sacs

Lysosomes are membranous sacs containing digestive enzymes

Packages secretions; manufactures lysosomes

Lysosomes release enzymes to breakdown proteins and other materials, including ingested
bacteria; play role in cell death Vacuoles Membranous sacs Contain ingested materials or
cellular secretions of waste Centrioles Non membranous; pair of hallow cylinders Mitotic
spindle forms between these structures during mitosis Nucleus Nucleus Large spherical
structure surrounded by a double nuclear membrane; contains nucleolus and chromosomes
Control center of the cell; contains the chromosomes Nucleolus Non membranous; rounded
granular body within nucleus; consist of RNA and protein Assembles ribosome; may have other
functions Chromosomes Non membranous; long threadlike structures composed of DNA and
proteins Contain the genes (hereditary units) that govern the structure and activity of the cell

Prokaryotes Microorganisms (Bacteria and Archaea)

The prokaryotes are a group of organisms that lack (=pro “before”) a cell nucleus (= karyon), or
any other membrane-bound organelles. They differ from the eukaryotes, which have a cell
nucleus. They multiply by binary fission.

The prokaryotes are divided into two domains: the bacteria and the archaea (the origin of
Cyanobacteria).
Prokaryotes or Bacteria cell structure

Capsule  It is a layer that lies outside the cell wall of bacteria. It is a well organized layer, not
easily washed off, and contributes to pathogenicity.  Function is to protect the bacteria  It is a
thick wall which consists of two-thirds (2/3) sugar (polysaccharides) and a third (1/3) protein 
The cell capsule is a very large structure of some prokaryotic cells  Attachment to surfaces;
protection against phagocytic engulfment, occasionally killing or digestion; reserve of nutrients
or protection against desiccation  An example of encapsulated bacteria are Mycobacteria

Cell Wall  Acts as another layer of protection for the bacteria  Helps bacteria to maintain their
shape  Helps prevent cell lysis caused by increased osmotic pressure  Participates in cell
division  Composition is similar to that of the capsule  Bacterial cell walls are made of
peptidoglycan (also called murein), which is made from polysaccharide chains and peptides.

Composition of the cell wall makes the bacteria either GRAM (-) or GRAM (+)  Gram-positive
bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acids. 
Gram-negative bacteria have a relatively thin cell wall consisting of a few layers of
peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and
lipoproteins.  These differences in structure can produce differences in antibiotic susceptibility
Cell Membrane  Consists of carbohydrate, protein, and phospholipids, called lipid bilayer 
Contains pores which allows nutrition to enter and waste to exit by active transport mechanism
 Helps in the cell division process, respiration, secretion of extracellular enzymes and toxins

Spores  A dormant non-reproductive body formed by certain bacteria, fungi, algae, and non-
flouring plants. Spores are formed in response to adverse environmental conditions and are
resistant to disinfectant, boiling, refrigeration and drying  They can revert to the vegetative
active state when conditions become favorable (germinate)

Flagella and Basal Body  Main function is to move bacteria (locomotion)  Basal body is
attached to the cell wall

Mesosome  Is a folded cell membrane  Only opens during cell division to give bacteria a
bigger surface area  Bacteria divide every 20 minutes

Ribosome  Function is for protein synthesis from all amino acids  The amount of ribosome
indicates the speed of metabolism  Ribosomes are made from complexes of RNA and protein.
Ribosomes are divided into two subunits, one larger than the other

Inclusion Granules  Function is to reserve energy and nutrients  Provide bacteria nutrition
during dormant period  The size of those granules can increase in favorable environment and
decrease when conditions are adverse

Nucleoid Function is to control all the bacteria’s activity  The nucleoid is a region of cytoplasm
where the chromosomal DNA (Deoxyribonucleic acid) is located  It is not a membrane bound
nucleus, but simply an area of the cytoplasm where the strands of DNA are found  Most
bacteria have a single, circular chromosome that is responsible for replication, although a few
species do have two or more. Smaller circular auxiliary DNA strands, called plasmids, are also
found in the cytoplasm.
Two (2) types: Ordinary Pili Helps bacteria to float and get transferred Sex Pili  An old
mycobacterium will attach itself to a younger one and transform its genetic material to build a
much stronger and resistant bacteria. TB resistance may be acquired this way

Classification of Bacteria (Based on shape)

 Different types of bacteria are classified according to shape  The predominant morphologic
shapes are:
Bacillus

 Rod-shaped bacteria  Under stressful environmental conditions some bacilli produce oval
endospores that can stay dormant for extended periods.  Two Bacillus species are considered
medically significant: B. anthracis, which causes anthrax, and B. cereus, which causes a
foodborne illness  Clostridium also makes endospores

Spiral
 Gram Negative Spiral shaped with twisting motility  Example is Treponema pallidum which
causes syphilis  Spirillum is a thick rigid spiral  Spirochete is a thin, flexible spiral

Vibrio

Types of Bacillus Bacteria

Diplobacilli

Streptobacilli

 Shaped like a comma  Example is the cholera bacteria  They are motile organisms, using a
single polar flagellum to travel.  Many Vibrio are pathogenic to humans.  V. vulnificus is
responsible for 95% of seafood-related deaths.  Another major disease caused by Vibrio
species is cholera, which occurs when V. cholerae colonizes the small intestine and releases
enterotoxins

Classification of Bacteria (based on Oxygen requirement)

DEFINITIONS:

Aerobic bacteria, also called aerobes, can live and survive only in an oxygenated environment.
They use oxygen for complete metabolic breakdown of food molecules

Anaerobic bacteria or anaerobes grow in the absence of oxygen. Such bacteria most commonly
infect deep tissues, internal organs, and deep lacerations (must live in a carbon dioxide rich
environment).

Obligate aerobes require oxygen for the oxidation of substrates (such as sugar and fat) in order
to derive energy

Microaerophiles are organisms that may use oxygen, but only at low concentrations

Facultative aerobes are microorganisms that can live in the presence or absence of oxygen

Obligate anaerobes do not require oxygen for the breakdown of substrates. Furthermore, they
get killed in the presence of oxygen.

Facultative anaerobes, oxygen is not the limiting factor because they can survive both in the
presence and absence of oxygen

Aerotolerant organisms, which cannot use oxygen for growth, but tolerate the presence of it

Nutritional Requirements

 Autotrophic bacteria are able to live in a strictly inorganic environment, i.e., iron, sulphur,
ammonia, nitrite, hydrogen

 Heterotrophic bacteria require an organic environment - i.e., carbohydrates, peptone. The

majority of medically important bacteria are heterotrophs.