MICROBIO

(MIDTERMS REVIEWER)

LECTURE 2: Continuation…
MAGNIFICATION vs. RESOLUTION

 High magnification without high resolution may make very small microbes visible, it will not allow
the observer to distinguish between microbes or sub-cellular parts of a microbe.
 Microbiologists depend more on resolution, as they want to be able to determine differences
between microbes or parts of microbes

FEATURES OF A GOOD MICROSCOPE

1. Adequate magnifying power
2. Provide good contrast
3. Possess high resolving power
4. Serves its purpose

PREPARING SPECIMENS TO BE OBSERVED

1. Wet mount
 simplest type of preparation
 the specimen is placed on the slide in a drop of liquid
 Sometimes the liquid used is simply water, but often stains are added to enhance contrast
 wet mount tends to dry out quickly under the heat of the microscope light
 useful for short-term observation only
 Wet-mounted preparations are used primarily to detect microbial motility rapidly
 the preparation tends to dry up more quickly, even when sealed
2. Hanging drop
 more complex technique
 allows for longer-term observation because the drop does not dry up quickly and more
reliable observation of motility
 usually performed without the addition of any stains; the organisms can be difficult to see.
 The hanging drop and wet mount techniques allow for observation of living organisms.
 Primarily, the method is used to determine whether an organism is motile
 Vibration of the cell is caused by the cell colliding with water molecules
 True motility allows the cell to move in different directions and across larger areas

3. Stained Preparation
 3 Basic Steps
 Smear preparation
 Smear is a preparation process where a specimen is spread on a slide
 Fixation
 Heat fixation (direct flame or steam)
 Chemical fixation (alcohols)
 “fixing” or fixation of a sample refers to the process of attaching
cells to a slide
 often achieved either by heating (heat fixing) or chemically treating
the specimen
 Purpose of fixation
i. Kills the cells
ii. Makes the cells sticky so they adhere to the slide
iii. Increases apparent diameter of cells
 iv. In addition to attaching the specimen to the slide, fixation
also kills microorganisms in the specimen, stopping their
movement and metabolism while preserving the integrity of
their cellular components for observation.
 Staining
 almost always applied to color certain features of a specimen before
examining it under a light microscope
 Stains, or dyes, contain salts made up of a positive ion and a negative ion.
 the positive or the negative ion may be the chromophore (the
colored ion); the other, uncolored ion is called the counterion.
 If the chromophore is the positively charged ion, the stain is classified as
a basic dye
 if the negative ion is the chromophore, the stain is considered an acidic
dye
 In most cases, it is preferable to use a positive stain, a dye that will be
absorbed by the cells or organisms being observed, adding color to
objects of interest to make them stand out against the background.
 commonly used basic dyes such as basic fuchsin, crystal violet,
malachite green, methylene blue, and safranin typically serve
as positive stains
 However, there are scenarios in which it is advantageous to use a
negative stain, which is absorbed by the background but not by the cells
or organisms in the specimen
 Negative staining produces an outline or silhouette of the
organisms against a colorful background
 Commonly used acidic dyes include acid fuchsin, eosin, and rose
bengal.
 Some staining techniques involve the application of only one dye to the
sample; others require more than one dye.

Simple staining
o Single dye is used to emphasize structures in the specimen
o A simple stain will generally make all the organisms in a sample
appear to be the same color, even if the sample contains more
than one type of organism.
Differential Staining
o In contrast, differential staining distinguishes organisms based
on their interactions with multiple stains
o two organisms in a differentially stained sample may appear to be
different colors
o Differential staining techniques commonly used in clinical
settings (include Gram staining, acid-fast staining, endospore
staining, flagella staining, and capsule staining).
Gram Staining
o The Gram stain procedure is a differential staining procedure
that involves multiple steps
o It was developed by Danish microbiologist Hans Christian Gram
o effective method to distinguish between bacteria with different
types of cell walls
 o even today it remains one of the most frequently used staining
techniques.
o Gram-staining is a differential staining technique that uses a
primary stain and a secondary counterstain to distinguish
between gram-positive and gram-negative bacteria

 The steps of the Gram stain procedure are listed and

illustrated in Table below.
o 1. First, crystal violet, a primary stain, is applied
to a heat-fixed smear, giving all the cells a purple
color.
o 2. Next, Gram’s iodine, a mordant, is added. A
mordant is a substance used to set or stabilize
stains or dyes; in this case, Gram’s iodine acts like a
trapping agent that complexes with the crystal
violet, making the crystal violet–iodine complex
clump and stay contained in thick layers of
peptidoglycan in the cell walls.
o 3. Next, a decolorizing agent is added, usually
ethanol or an acetone/ethanol solution. Cells that
have thick peptidoglycan layers in their cell walls
are much less affected by the decolorizing agent;
they generally retain the crystal violet dye and
remain purple. However, the decolorizing agent
more easily washes the dye out of cells with thinner
peptidoglycan layers, making them again colorless.
o 4. Finally, a secondary counterstain, usually
safranin, is added. This stains the decolorized cells
pink and is less noticeable in the cells that still
contain the crystal violet dye.

LECTURE 3:
Isolation and Cultivation of Microorganism

 To study the specific role played by a specific microorganism in its environment, one must
isolate the same in pure culture
Pure culture
o a culture which contains a single species of microorganisms
o a population of cells arising from a single cell
Isolation
o The process of obtaining a pure culture by separating one species of microbe
from a mixture of other species

Cultivation
o increasing the population of microorganisms by providing their nutritional and
physical requirements

ISOLATION OF MICROORGANISMS
 A culture which contains just one species of microorganism is called a pure culture.
  The process of obtaining a pure culture by separating one species of microbe from a mixture of other
species, is known as isolation of the organisms.

Pure cultures are important in microbiology for the following reasons:

■ Once purified, the isolated species can then be cultivated with the knowledge that only the desired
microorganism is being grown.
■ A pure culture can be correctly identified for accurate studying and testing and diagnosis in a clinical
environment.
■ Testing/experimenting with a pure culture ensures that the same results can be achieved regardless of
how many times the test is repeated.

Isolation Techniques
1. Plating
2. Enriched procedure
3. Dilution
4. Single cell technique

1. Plating
 This is most widely used method of isolation
 The technique consists of pouring a suitable sterile medium into sterile Petri plate and allowing the
medium to solidify

1.1 Streak Plate Method

 streaked back and forth across the surface of agar until about one third of the diameter
1.2 Pour Plate Method
 The bacterial culture and liquid agar medium are mixed together
 After mixing the medium, the medium containing the culture poured into sterilized Petridishes
(Petriplates), allowed solidifying and then incubated.

Disadvantages of Pour plate method:

 The microorganisms are trapped beneath the surface medium when it solidifies
 This method is tedious, time consuming and requires skill.
 The microorganisms are subjected to hot shock because liquid medium is maintained at 45°C
temperature
 This method is unsuitable for isolation of psychrophile bacteria

1.3 Spread Plate Method

 The spread plate method is a technique to plate a liquid sample containing bacteria so that the
bacteria are easy to count and isolate.
 culture technique is among the most widely used culture technique for isolating the bacteria

Advantages of spread plate method:

 It is a simple method
 In this method only surface colonies are formed.
 Microorganisms are not exposed to higher temperature

2. Enrichment culture
 isolation of specific types of microorganisms by a combination of nutrient and physical conditions

3. Serial dilution
  A serial dilution is a series of sequential dilutions used to reduce a dense culture of cells to a more
usable concentration.
 This method is used for the microorganisms which cannot be easily isolated by streaking or plating
method.
 Each dilution will reduce the concentration of bacteria by a specific amount.

4. Single-cell isolation technique

 Uses a micropipette or microprobe to physically pick a single cell and transfer it on an agar medium
 This is one of the most ideal and difficult method of securing pure culture

CULTIVATION OF MICROORGANISMS
 The various mixtures of nutritive substances used for the laboratory cultivation of microorganisms
are collectively known as culture media.
 Culture media must contain all the essential nutrients required by the organism for its growth and
reproduction.
 The culture media serve as soil in which bacteria are planted for the purpose of study.

A culture medium must contain:

 Carbohydrate (sugar) – source of hydrogen, carbon and chemical-bond energy
 Nitrate ions or Ammonium ions – source of nitrogen
 Sodium / Magnesium sulphate – minerals
 Micronutrients
 Growth factors, vitamins, & certain amino acids

Importance of Culture Media

o To obtain pure cultures
o To grow and count microbial cells
o To cultivate and select microorganisms

Common Ingredients of Culture Media

1. Water
o It is essential for existence of living cells. They act as source of hydrogen and oxygen.
2. Peptone
o Golden granular hygroscopic powder which are obtained from meat, casein fibrin or soya bean flour.
Function: nitrogen source, carbon source, buffers
o Main pH level – 7.3 to 7.4 –> buffer (need to be neutral) – 7.1 to 7.5
3. Meat Extract
o It contains protein degradation products, carbohydrates, inorganic salts, enzymes, excites and
growth factors that are rich in vitamin B complex.
 Function: Source of growth factors, inorganic salts etc.
4. Yeast Extract
o It contains proteins, amino acids, growth factors (Vitamin B), Carbohydrates and inorganic salts like
potassium and phosphates.
 Functions: Source of growth factors and hence excellent stimulators of growth. It can be used
as suitable for meat extract.
5. Electrolyte
o Mainly used are sodium chloride or other electrolytes.
 Functions: Essential to maintain the osmotic pressure
o Too much salt will leads to no growing will occur in your culture media
6. Agar
o Dried mucilaginous substance obtained from gelidium species and other algae available as long
shield or in powder form; contains mainly long chain polysaccharides, protein like material and
inorganic salts.
 Functions: it melts at 98°C and solidifies at 42°C, hence used as solidifying agent.
7. Fermentable Compounds
o Mainly used are sugars, alcohols etc.
 Function: Act as source of energy, fermentation reactions are helpful in the identification and
classification of organisms.
o Pag nagchange ang color ng colonies, it means nag-undergo yon ng fermentation; pag white lang,
hindi nag-undergo
8. Buffers
o Carbonates and phosphates are used as buffer. Function: To resist change in pH of the medium

Types of Culture Media according to physical state:

1. Liquid (broth) - no solidifying agent
a. No agar added
b. Nutrient broth – consists of meat extract, peptone, NaCl
i. Usually yellow ang color bcoz of the peptone
c. Beef infusions – also called infusion broth
2. Semi-solid – with 0.1 to 0.5% solidifying agent
a. Used for determining motility
b. Useful sa pag-oobserve ng mga bacteria na nabubuhay kahit na lesser ang oxygen sa
environment
c. Red algae - agar
3. Solid – with 1.5 to 2.0% solidifying agent (ex. agar or gelatin)
a. Used for colony characterization and classification (such as structure)
b. Every colony has a different characteristics

Categories of media according to purpose or application:

1. General
2. Enrichment
3. Selective
4. Differential and
5. Propagation

1. General Media:
o General purpose media that will support the growth of a wide variety of bacteria include nutrient
agar, tryptic soy agar, and brain heart infusion agar.
 pH level (7.3-7.4)
 usually used for culturing your media
2. Enrichment media:
o They are prepared with ingredients that will enhance the growth of certain microbes.
 Addetives:
 Blood
 Syrum
 Blood agar or chocolate agar
o Enrichment media encourage the growth of the suspected pathogen so that it will become the most
pre-dominant type of microbe in the culture.
3. Selective media:
 o They are prepared with ingredients that inhibit the growth of unwanted microbes which might be in
the specimen.
 Addetives:
 Antibiotics or other chemicals (salt)
 Manitol salt agar

4. Differential media:
o They are designed to differentiate among microbes. Different bacterial species may produce
dissimilar colony colours when grown on differential agar.
 May nilalagay na certain type of stain (crystal violet or iodine solution)
 Sobrang daming colors
5. Propagation media:
o They are used to propagate, or keep microbes growing for a long lime. Samples grown on these
media may be taken for analysis.
 Common ingredient ay nutrient broth and agar
 Mas madaling makakapagpadami pag liquid and gamit kaysa solid na agar

COMMON MEDIA
 Tryptic Soy Agar (TSA) – a general media
o They can allow different kinds of media to grow
o Common na gingamit sa lab
o Pastigeous - inaallow din nito ang growth ng type ng bacteria na may certain diet na
kinakailangan para maggrow
 ex. ni-ceria (need ng blood and vit) helicobacteria (need ng mataas ng CO2 level)
o non-pastigeous - pede silang maggrow kahit di mo lagyan ng certain diet ang agar
 (e.coli at streptococcus)
o oks lang kahit meat extract and agar lang
 Chocolate/Blood agar
o need lagyan ng red blood cells (sheep blood)
o nagsubject sya sa lysis (kaya tinawag na choco)
o essentially chocolate agar is blood agar kaso tinatawag lang na chocolate agar pag pinainit
(ang tawag don ay lysis)
o lysis ay process ng pag-patay sa RBC
 Thayer-Martin Agar
o improve (peptone, hemoglobin, constarch and 2-3% agar)
o ang naggrow dito ay niceria at meningitidis (all species ng niceria)

 EMB (selective media)

o gram negative bacteria lang ang naggrow dito
 (ex. usually pathogens ang ginogrow dito)
 MacConkey - (selective)
o gram negative lang din ang naggrow na bacteria (consists of salts, crystal violet) also called as
lactose agar
o (pag transparent or puti yung colony- di sila nag undergo ng lactose fermentation; vv. = pink
or light red - merong lactose fermentation)
 MSA
o sobrang daming NaCl (staphilococcus ang usually naggrow dito)
o resistant sila (staphilococcus) sa salt concentration
  Blood
o contentrated blood (usually used for pastigeous bacteria)
 (components: peptone, NaCl)
 Lowenstein (Differential)
o used of genus of microbacterium (distinguishing ingr. = nilalagyan ng itlog for protein and
fatty acids)
o isa tong differential media (stain = malakyte green) may potato flour tong agar na to for
carbohydrates
 Hektoen
o usually pathogens ang ginagamitan nito
 (specifically the shigella and salmonella)

 Medium containing heat- sensitive substances are sterilized either by filtering the solution at room
temperature, using bacteria-proof filter or by a process called Tyndallization

Lesson 4:
Control and Destruction of Microorganisms

Reasons for controlling microbial growth:

1. To prevent/limit spoilage or destruction of valuable substances/commodities
2. To prevent infections
3. To prevent contamination of the cultures, the person and the environment

Microorganisms are controlled by means of physical agents and chemical agents.

o Physical agents
 include such methods of control as high or low temperature, desiccation, osmotic pressure,
radiation, and filtration
o Chemical agents
 refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial
chemicals

Microbial control can be achieved through:

1. Inhibition of microbial growth (prevent from growing, multiplying)
2. Destruction of the microorganism

Basic terms used in discussing the control of microorganisms include:

 Sterilization
o Sterilization is the process of destruction, removal elimination and inactivation of all forms of
microbes from culture media and body surfaces etc.
 Disinfection
o Disinfection is the elimination of microorganisms from inanimate objects or surfaces.
 Decontamination
o Decontamination is the treatment of an object or inanimate surface to make it safe to handle.
Sterilization and disinfection are forms of decontamination.
 Disinfectant
o A disinfectant is an agent used to disinfect inanimate objects but generally too toxic to use on
human tissues.
 Antiseptic
 o An antiseptic is an agent that kills or inhibits growth of microbes but is safe to use on human
tissue.
 Sanitizer
o A sanitizer is an agent that reduces, but may not eliminate, microbial numbers to a safe level.
 Cidal
o An agent that is cidal in action will kill microorganisms and viruses.
 Static
o An agent that is static in action will inhibit the growth of microorganisms.
PHYSICAL AGENTS

I. TEMPERATURE
 Temperatures below the minimum usually have a static action on microorganisms.
 They inhibit microbial growth by slowing down metabolism but do not necessarily kill the
organism.
 Temperatures above the maximum usually have a cidal action, since they denature microbial
enzymes and other proteins.

i. High Temperature
 Vegetative microorganisms can generally be killed at temperatures from 50°C to 70°C
with moist heat.
 Bacterial endospores, however, are very resistant to heat and extended exposure to
much higher temperature is necessary for their destruction.
 High temperature may be applied as either (a)moist heat or (b)dry heat.
o (a.) Moist heat
 Moist heat is generally more effective than dry heat for killing
microorganisms because of its ability to penetrate microbial cells.
 Moist heat kills microorganisms by denaturing their proteins
(causes proteins and enzymes to lose their three-dimensional
functional shape).
 Autoclaving (temperature of 121° C)
 Autoclaving employs steam under pressure.
 Boiling water
 Boiling water (100°C) will generally kill vegetative
cells after about 10 minutes of exposure.
 However, certain viruses, such as the hepatitis
viruses, may survive exposure to boiling water for
up to 30 minutes
o (b.) Dry heat
 Hot air sterilization
 They are generally used only for sterilizing
glassware and metal instruments.
 Incineration
 Incinerators are used to destroy disposable or
expendable materials by burning.
Low temperature
 Low temperature inhibits microbial
growth by slowing down microbial
metabolism.
  Refrigeration at 5°C slows the
growth of microorganisms and keeps
food fresh for a few days
 Freezing at -10°C stops microbial
growth, but generally does not kill
microorganisms, and keeps food
fresh for several months.
 Dessication
 Desiccation, or drying, (sun, air, oven) generally has
a static effect on microorganisms.
 Lack of water inhibits the action of microbial
enzymes.
 Radiation
Ultraviolet Radiation
 An important consideration when
using UV light is that it has very poor
penetrating power.
 Only microorganisms on the surface
of a material that are exposed
directly to the radiation are
susceptible to destruction.
 UV light can also damage the eyes,
cause burns, and cause mutation in
cells of the skin.
Ionizing Radiation
 Ionizing radiation, such as X-rays and
gamma rays, has much more energy
and penetrating power than
ultraviolet radiation.
 It is often used to sterilize
pharmaceuticals and disposable
medical supplies
 It can also be used to retard spoilage
in seafoods, meats, poultry, and
fruits.
 Filtration
 Microbiological membrane filters provide a useful
way of sterilizing materials such as vaccines,
antibiotic solutions, animal sera, etc. that may be
damaged or denatured by high temperatures or
chemical agents.
CHEMICAL AGENTS

DISINFECTANTS, ANTISEPTICS, AND SANITIZERS

 Disinfection is the elimination of microorganisms from inanimate objects or surfaces
 Usually unreliable for the destruction of all life forms.

There are several factors which influence the antimicrobial action of disinfectants and antiseptics,
including:
 The concentration of the chemical agent.
  The temperature at which the agent is being used.
 The kinds of microorganism present.
 The number of microorganism present.
 The nature of the material bearing the microorganisms.

EFFECTIVENESS OF HAND WASHING

 It is routine practice to wash the hands prior to and after examining a patient and to do a complete
regimented surgical scrub prior to going into the operating room.
 Actual sterilization of the hands is not possible since microorganisms live not only on the surface of
the skin but also in deeper skin layers, in ducts of sweat glands, and around hair follicles.

Lesson 5:
Classification of Microorganism

Microbes and the World

 Each type has a characteristic cellular composition, morphology, mean of locomotion, and
reproduction.
 Microorganisms are beneficial in producing oxygen, decomposing organic material, providing
nutrients for plants, and maintaining human health, but some can be pathogenic and cause diseases
in plants and humans.

Environmental Diversity of Microbe

 Microbes live in every kind of habitat (terrestrial, aquatic, atmospheric, or living host) and their
presence invariably affects the environment in which they grow.

BACTERIA
 The cells are described as prokaryotic because they lack a nucleus.
 They exist in shapes: bacillus (rod shape), coccus (spherical shape), spirilla (spiral shape).
 Most bacteria have a peptidoglycan cell wall; they divide by binary fission; and they may possess
flagella for motility.
 According to the way their cell wall structure stains, bacteria can be classified as either Gram-
positive or Gram-negative when using the Gram staining.
 Aerobic (living in the presence of oxygen), anaerobic (living without oxygen), and facultative
anaerobes (can live in both environments).
 Autotrophs make their own food by using the energy of sunlight or chemical reactions, in which case
they are called chemoautotrophs.
 Heterotrophs obtain their energy by consuming other organisms.
 Bacteria that use decaying life forms as a source of energy are called saprophytes

ARCHAEA
 Archaea or Archaebacteria differ from true bacteria in their cell wall structure and lack
peptidoglycans.
 They are prokaryotic cells with avidity to extreme environmental conditions.
 Groups: Methanogens (methane-producing organisms), halophiles (Archaeans that live in salty
environments), thermophiles (Archaean’s that live at extremely hot temperatures), and
psychrophiles (cold-temperature Archaeans)

FUNGI
 Fungi (mushroom, molds, and yeasts) are eukaryotic cells (with a true nucleus).
  Most fungi are multicellular, and their cell wall is composed of chitin.
 They obtain nutrients by absorbing organic material from their environment (decomposers),
through symbiotic relationships with plants (symbionts), or harmful relationships with a host
(parasites).
 They form characteristic filamentous tubes called hyphae that help absorb material. The collection of
hyphae is called mycelium.
 Fungi reproduce by releasing spores.

PROTOZOA
 Protozoa are unicellular aerobic eukaryotes.
 They have a nucleus, complex organelles, and obtain nourishment by absorption or ingestion
through specialized structures.
 They make up the largest group of organisms in the world in terms of numbers, biomass, and
diversity.
 Their cell walls are made up of cellulose.
 Protozoa have been traditionally divided based on their mode of locomotion:
 Groups: Flagellates produce their own food and use their whip-like structure to propel forward,
ciliates have tiny hair that beat to produce movement, amoeboid have false feet or pseudopodia used
for feeding and locomotion, and sporozoans are non-motile.
 They also have different means of nutrition, which groups them as autotrophs or heterotrophs.

ALGAE
 Algae, also called cyanobacteria or blue-green algae, are unicellular or multicellular eukaryotes that
obtain nourishment by photosynthesis.
 They live in water, damp soil, and rocks and produce oxygen and carbohydrates used by other
organisms
 It is believed that cyanobacteria are the origins of green land plants.

VIRUSES
 Viruses are non-cellular entities that consist of a nucleic acid core (DNA or RNA) surrounded by a
protein coat.
 Although viruses are classified as microorganisms, they are not considered living organisms.
 Viruses often infest prokaryotic and eukaryotic cells causing diseases.
 The domain was proposed by the microbiologist and physicist Carl Woese in 1978 and is based on
identifying similarities in ribosomal RNA sequences of microorganisms.

MAJOR CHARACTERISTICS USED IN TAXONOMY

A. CULTURAL
▪ refer to the nutrients required for growth and the physical conditions of an environment that
will favor growth.

CULTURAL CHARACTERISTICS:
 Temperature requirement (minimum, maximum, optimum)
 Psychrophile
o Below 0˚C up to 20˚C
o Arctic, Antarctic, some have been on trapped ice
o Examples: Listeria monocytogenes, Arthobacter, Flavobacterium
micrococcus
  Mesophile
o 15˚C - 40˚C
o Involved in food contamination
o Examples: E.coli, streptococcus, Thiobacillus novellus, Staphylococcus
aureus, Streptococcus pneumonia
 Thermophile
o Above 113˚C
o Hot springs, hydrothermal vents (under the ocean)
o Examples: Pyrolobus fumarii, Pyrococcus abyssi
 Oxygen Requirement
 Aerobic
o Examples: Pseudomonas aeruginosa, Nocardia, Mycobacterium t.,
Bacillus sp.
 Anaerobic
o Examples: Actinomyces, Clostridium
 Facultative anaerobes
o Examples: Staphylococci, Enterobacteriae, Lactococcus s.

B. MORPHOLOGICAL
▪ Morphology is easy to study and analyze both eukaryotic and prokaryotic microorganisms.

Unusually shaped bacteria

▪ Fruiting: Myxobacter
▪ Stalked: Caulobacter
▪ Budding: Hyphomicrobium
▪ Filamentous: Cyanobacteria, Actinomycetes
▪ Pleomorphic: Rhizobium, Mycoplasms (wall-less)
▪ Fusiform: Fusobacterium

CELL ARRANGEMENT
 Diplococcus/diplococci – in pairs
 Streptococcus/streptococci – in chains
 Staphylococcus/staphylococci – forms irregular mass resembling a cluster of grapes
 Tetrads – 4 cells
 Sarcina – packets of 8 cells