UNIT II B- PROCESSES AND PURPOSES OF THE SIX TYPES OF PROCEDURES THAT ARE USED IN HANDLING, MAINTAINING, AND STUDYING MICROORGANISMS

Question 1

Inoculation

Answer 1

Ethmology: inoculare – ‘to graft’
- introduction of microorganisms into a growth
medium where they can grow and
reproduce - In Immunology, inoculation is the
introduction of an antigenic substance like
antigens that induce an immune response or
a vaccine (weakened, dead, inactivated
pathogens that are inoculated into the body
in order to induce immunity to that
pathogens)
- In Microbiology, inoculation is the
introduction of microorganisms into a culture
where they are able to grow and reproduce

Question 2

Agar plates

Answer 2

most common media used in labs for
growing bacteria and other
microorganisms
- combined with nutrients necessary for
bacterial growth and poured into circular
plates called Petri dishes where the
agar solution solidifies
- A solution containing the
microorganisms being studied is
inoculated unto these plates usually via
streaking
- Usually, a small streaking loop is used in
dipping it into a solution containing
bacterial cells and is used to streak unto
the plate

Question 3

Liquid media suspensions

Answer 3

• grow bacteria
• a single cultured bacteria is added to a
  small solution mixed and pipetted into
  the liquid media

Question 4

History: Smallpox

Answer 4

Smallpox afflicted humans since 10,000 BC
- Evidence shown through the faces of
Egyptian mummies from the 1500-1000 BC
- “speckled monster

Question 5

History: Smallpox: Inoculation

Answer 5

• Originated in China as early as 1567
• Before: variolation
• Performed against smallpox
• Performed by blowing small parts of the
  infectious smallpox material up the nose to
  be inoculated; the process called
  Insufflation
• The infectious material either ground down
  into powder or the pus from pustules was
  collected and placed up the nose

Question 6

History: Smallpox: Inoculation: Location

Answer 6

• India may be the origin of inoculation - Involves a sharp needle being dipped into
  infectious pustules and placed into skin of
  recipients
• This inoculation process was used in Bengal
  and Bangladesh
• In Europe, inoculation against smallpox
  was carried out in the 18th century using a
  surgical knife, lancet
• with a portion of pustule in the skin, a
  disinfected lancet was inserted in
  subcutaneous layer
  Lady Mary Montague – suffered from
  smallpox and got scars due to it
• In 1721, variolation came to America
• In 1777, all soldiers were variolated before
  going to their duties

Question 7

Variations of smallpox: Variola major

Answer 7

Characteristics: (1) ordinary, 
(2) modified, (3) flat, and (4) 
hemorrhagic
NOTE: Flat and hemorrhagic 
smallpox were more closely 
associated with death.

Question 8

Other variation of smallpox

Answer 8

variola minor

Question 9

History: Smallpox: Edward Jenner

Answer 9

• Edward Jenner interested in the relationship
  of cow pox and smallpox found in
  dairymaids in 1796
• 1796 - dairymaid had cowpox lesions on her
  hands
• inoculated a small boy of 8 years old
• became mildly unwell with a fever but
  recovered
• he inoculated the boy again-tissue from
  smallpox lesions
• boy never developed any disease
• proved that the inoculation protected the
  boy from smallpox

Question 10

Variolation

Answer 10

-replaced by vaccination
– Variolation is illegal in England in 1840
- – obsolete method in immunizing
patients by infecting them with substance from the
pustules of patients with a mild form of the disease.

Question 11

How to inoculate in culture media?

Answer 11

• inoculation involves adding a portion of
  specimen to the medium
• inoculation is accomplished using a sterile
  inoculation loop
• can be placed in Media in petri dishes/Slope
  media (Agar Slants)/Inoculation of stab
  media(deeps)/Inoculation of fluid media
• Stab media – uses a needle

Question 12

Aseptic techniques

Answer 12

- performed under sterile conditions to 
prevent any contamination
1. Use of Bunsen burner or spirit lamp--
sterilize wire loops/inoculating needles
2. Flame the necks of specimen bottles, 
culture tubes

Question 13

Biological Safety Cabinets

Answer 13

• used to protect personnel against
  biohazardous or infectious agents
• help maintain quality control of the material
  being worked
• referred to as a laminar flow or tissue
  culture hood
• primary engineering control used to protect
  personnel against infectious agents and
  help maintain quality control on the material
  being worked with as it filters the in-flow and
  exhaust air
• protect against exposure to particulate or
  aerosols
• a portion of the air is circulated back into the
  lab through its exhaust heap filter – purifying
  the air from potentially infectious aerosols

Question 14

Plating

Answer 14

– inoculating media in a Petri dish

Question 15

Transfer techniques

Answer 15

• Agar plate-to-Agar Slant
• Broth-to-Broth Transfer
• Broth-to-Slant Transfer

Question 16

Incubation

Answer 16

• to promote growth of microbes
• atmosphere
• temperature
• duration
• microbes grow and multiply, producing
  visible growth in the media

Question 17

Isolation

Answer 17

if an individual cell is separated from other
cells in a nutrient surface, it will form a
colony
Colony – macroscopic cluster of cells
appearing in a solid medium arising from the
multiplication of a single cell

Question 18

Isolation of Pure Culture methods

Answer 18

• Streak Plate
• Pour Plate
• Spread Plate and dilution technique

Question 19

Inspection

Answer 19

Observation; macroscopic and microscopic
Pure culture: Grows only single known 
species of microorganisms
Mixed culture: Hold two or more identified 
species or microorganisms
Contaminated 
culture:
Once pure or mixed culture that 
has unwanted microbes 
growing

Question 20

Information gathering

Answer 20

• Collection of data to help support a final
  determination of the types of the microbes
  present
• Can be accomplished using variety of
  schemes
• “What data to collect?” - Analysis can be quantitative (numerical
  information) or qualitative (yes or no for an
  answer)
  Qualitative: growth of a bacterial sample on a
  solid growth medium to assess whether the
  bacteria of the sample are living or dead
  Quantitative: calculate the actual number of
  living bacteria in a sample

Question 21

Identification

Answer 21

• Identify samples like pure colonies
• Essential in analysis
• Identifying a novel bacteria isolate
  Bacteria – identified routinely by:
  • Morphological tests
  • Biochemical tests
  • Serotyping
  • Antibiotic inhibition patterns
  • Molecular techniques permit species to be
  identified by their genetic sequences;
  delineate and identify its true identity down
  to species level
  Books and Manuals Useful in Identification
  • Bergey’s Manual of Systematics
• Main resource in determining the
  identity of prokaryotic organisms
  using characterizing aspects
• Does not classify bacteria according
  to evolutionary relatedness
  • The Bergey’s manual of determinative
  bacteriology