Chapter 10 Culture media preparation, inoculation.ppt

Culture media are artificially prepared media containing the required nutrients used for propagation of micro organisms.

Once the bacteria are grown we can:
1. Identify them either by presumptive lab diagnosis like Gram
stain or by definitive lab diagnosis like biochemical test
2. Test the antimicrobial sensitivity of the bacteria (drug
testing). This helps to know whether the bacteria are
sensitive or resistant to known antimicrobial drugs.

1. CHAPTER X
PREPARATION OF CULTURE MEDIA,
INOCULATION AND IDENTIFICATION OF
BACTERIAL GROWTH CHARACTERISTICS
1

2. ACKNOWLEDGMENT
•ADDIS ABABA UNIVERSITY
•JIMMA UNIVERSITY
•HAWASSA UNIVERSITY
•HARAMAYA UNIVERSITY
•UNIVERSITY OF GONDAR
•AMERICAN SOCIETY OF CLINICAL PATHOLOGY
•CDC-Ethiopia
2

3. Learning objectives
At the end of this chapter, the student will be able :
1.List the common ingredients of culture media.
2.List the different types of culture media.
3.Discuss the different steps while preparing culture media.
4.Describe the storage methods for culture media.
5.Discuss the inoculation and aseptic techniques during
inoculation of culture media.
6.List the different inoculation techniques.
7.Discuss the culturing of anaerobes.
8.Perform quality control on culture media.
3

4. Culture media are artificially prepared media containing the
required nutrients used for propagation of micro organisms.
Once the bacteria are grown we can:
•1. Identify them either by presumptive lab diagnosis like Gram
stain or by definitive lab diagnosis like biochemical test
•2. Test the antimicrobial sensitivity of the bacteria (drug
testing). This helps to know whether the bacteria are
sensitive or resistant to known antimicrobial drugs.
4

5. Common ingredients of culture media
1. H
2O : is essential for growth of micro-organism and 80% of
bacterial cell is H
2O.
•It must be free of mineral salts which can inhibit bacterial
growth.
•Either distilled or deionised water should be used.
5

6. 2. Peptone: contains water soluble products obtained from break
down of animal or plant protein (contains free amino acids,
peptides, and proteoses (large sized peptides).
Eg. lean meat, heart meat, milk etc.
-It is the main source of nitrogen, some vitamins, carbohydrates,
nucleic acid fraction and minerals.
6

7. 3. Meat extract : this provides amino acids, minerals, and
essential growth factors .
4. Yeast extract : is a very good growth stimulant.
It provides amino acids, water soluble vitamins (vit.B),
inorganic slats, and etc.
5. Mineral salts :are essential for growth and for metabolism of
living organism.
E.g -SO4 -as a source of sulphur
-PO
4
2-
-as a source of phosphor
-Nacl -is the common ingredient and source of
sodium and chloride
-Other trace elements include Mg, Mn, etc
7

8. 6. Carbohydrates : simple and complex sugars are used:
•As energy and carbon source.
•For differentiation of bacteria.
Example:
-Sucrose in TCBS agar differentiates Vibrio species
-Lactose in MacConkey agar differentiates enterobacteria etc.
-Simple sugars includes glucose, maltose, sucrose, lactose,
pentose, hexose, galactose etc.
-The complex sugar include –polysaccharides, glycerol,
manitol etc.
8

9. 7. Solidifying agents: are agents which are used to solidify the
culture media.
A. Agar : is a commonly used solidifying agent of culture media.
•Is a mixture of two poly saccharide -agarose (70 -75%) &
agaropectin (20-25%).
•It is produced from sea weeds (Rhodophyceae) which form the
agarophyte group of marine algae .
9

10. •Is a good solidifying agent due to:
1. The protein is inert & undegradable by microorganism
2. Sol –gel transformation temperature is very high
i.e it solidifies at 45
o
C and changed to gel > 45
o
C
3. It can form gel (solidify) at 1.5% w/v concentration and
4. It forms semi solid gel at 0.4 –0.5% w/v concentration
Uses of agar
1.Solidify culture media
2.Provide calcium and organic ions for the bacteria
3.Used for adding heat sensitive ingredients such as blood and
serum while the agar is in gel phase at 45 –50
o
C
4.The agar is very transparent so that growth of colonies is
easily visible.
10

11. B. Gelatin –is sometimes used as solidifying agent but it has
many disadvantages like:
–The protein which are found in gelatin are susceptible to microbial
digestion
–Solid to gel transformation is very low which is 24
o
C, so it will become
changed to gel at 24
o
C & can not be used for bacteria which need
incubation above 25
o
C.
–15% w/v gelatin is required to solidify the culture.
8. Accessory growth factors
•Include vit B (thiamine, niacin, and biotin).These provide the
necessary condition for growth
9. Other ingredients include, blood, serum, egg yolk etc
depending on the organisms need.
11

12. Types of culture media
The main types of culture media are:
1. Basic media
2. Enriched or enrichment media
3. Selective media
4. Indicator (differential) media
5. Transport media
12

13. 1. Basic Media
•These are simple media that will support the growth of micro
organisms that do not have special nutritional requirements.
Example -Nutrient Agar
-Nutrient broth
Purposes of basic media
1. Are used in preparation of enriched media.
2. Are used to maintain stock culture.
3. For sub culturing pathogens from differential and selective
media prior to performing biochemical and serological
identification tests.
13

14. 2. Enriched/Enrichment media
These media are required for growth of organism with extra
nutritional requirements such as H. influenza, Neisseria spp,
and some streptococcus species.
An enriched medium increases the number of a pathogen by
containing all the necessary ingredients to promote its growth.
The media can be enriched with whole blood, lyzed blood,
serum, vitamins, and other growth factors.
E.g: -Blood Agar (contain whole blood)
-Chocolate agar (contain lyzed blood)
•Enrichment media: this term is usually applied to fluid
selective media E.g. Tryptosoya broth
Selenite F broth
14

15. 3. Selective media
•These are solid media which contain substances which inhibit the
growth of one organism to allow the growth of the other to be
more clearly demonstrated.
•The medium is made selective by incorporation of certain
inhibitory substances like bile salt, crystal violet, antibiotics, etc.
•E.g. Addition of crystal violet favors the growth of gram negative bacteria and slows
the growth of Gram positives.
•Selective medium is used when culturing a specimen form a site
having normal microbial flora to prevent unwanted contaminants
overgrowing a pathogen.
E.g.1. Thiosulphate citrate bile salt sucrose agar(TCBS) -is
alkaline medium and selective for V. cholera.
15

16. 2. Xylose Lysine Deoxy Cholate (XLD) agar -selective for
Salmonella and Shigella.
3. Modified New York City (MNYC) medium -Selective for
Neisseria gonorrhoeae.
4. Butzler medium -Selective for Campylobacter species.
5. Salmonella and Shigella agar (SSA) -Selective for Salmonella
and Shigella species.
16

17. 5. Differential (Indicator) media
•These are media to which dyes or other substances are added
to differentiate micro-organisms.
•Many differential media distinguish between bacteria by
incorporating an indicator which changes colour when acid is
produced following fermentation of a specific carbohydrate.
E.g. Mac Ckonkey agar -contain neutral red as an indicator
and lactose as carbohydrate. Lactose fermenting bacteria will
become pink and other bacteria become colourless.
17

18. Figure: differential media (Triple sugar iron/TSI agar media)
Figure: differential media (MacConkey agar media , pink colonies are
lactose fermenters)
18

19. 6. Transport media
•These are mostly semisolid media that contain ingredients to
prevent the overgrowth of commensals and ensure the survival
of aerobic and anaerobic pathogens when specimens cannot be
cultured immediately after collection.
•Their use is particularly important when transporting
microbiological specimens form health centres to the district
microbiological laboratory or regional public health
laboratory.
19

20. Example
1.Cary-Blair medium :–is used for preserving and transporting
enteric pathogens.
2.Amies transport medium:-is used for transportation of
gonococci.
20

21. Culture media can be classified by their consistency (form) as:
•Solid media
•Semi-solid media and
•Fluid media
A. Solid media
•Are solidified by agar
•Are used mainly in Petri dishes as plate cultures, in bottles or
tubes as stab (deeps) or slope cultures.
•The purpose of culturing on solid medium is principally to
isolate discrete colonies of each organism present in the
specimen. this will enable pure cultures to be produced for
identification and sensitivity testing.
NB: The colonial appearances and changes in solid media made by colonies may
provide valuable identification information
21

22. B. Semi solid media
•Are culture media prepared by adding small amount of agar
(0.4 -0.5% W/V) to a fluid medium.
•Semi solid media are used mainly as transport media, and for
motility and biochemical tests.
22

23. C. Fluid culture media
•Fluid media are most commonly used as enrichment when
organisms are likely to be few
E.g. blood culture
•Is also used for biochemical testing
E.g. Peptone water
23

24. 24

25. Preparation of culture media
For successful isolation of pathogens, culture media should be
prepared carefully following manufacturers instruction.
Most culture media are available commercially in ready-made
dehydrated powder form or ready made standardized culture
media (in packs)
25

26. Use of dehydrated culture media(powder form)
•Usually ready made standardized dehydrated media should be
used (i.e. all the ingredients are incorporated to ensure good
performance and reproducibility)than buying the individual
chemical constitutes.
•Dehydrated media is hygroscopic (i.e. it absorbs water), so
when exposed to moisture, it rapidly becomes unfit for use.
therefore to avoid this:
•Weigh the medium rapidly
•Tightly cap the bottle as soon as possible
•Do not return small amounts of medium to the stock and
•Store the media in the cool, dry place.
26

27. Use of ready made standardized culture media (in packs)
•These media are weighed ,suspended ,sterilized and packed in
small volumes (usually 15ml)
•These packed media only require boiling the sachet (packet)
in 95
o
C till it is changed to gel and dispense in Petri dish
aseptically.
27

28. Major procedures during Preparation of culture media
1. Weighing and dissolving of culture media
2. Sterilization and sterility testing
3. Addition of heat sensitive ingredients
4. pH testing of culture media
5. Dispensing of the culture media
6. Quality assurance of culture media
7. Storage of culture media
28

29. 1. Weighing and dissolving of culture media
•Use ingredients suitable for microbiological purpose
•Use clean glass ware, plastic or stainless steel equipment
•Use distilled water.
•Weigh accurately using a balance.
•Wear a facemask and glove while weighing and dissolving
toxic chemicals.
•Do not delay in making up the medium after weighing.
29

30. •Add powdered ingredients to distilled water and mix by
rotating or stirring the flask.
•Stir while heating if heating is required to dissolve the
medium.
•Autoclave the medium when the ingredients are dissolved.
•Over heating may result in:
-alteration of the nutritive value
-alteration of pH and
-alteration of gelling property
•Always autoclave media at the correct temperature and for the
time specified
30

31. 2. Sterilization and sterility testing
•Sterilization is required to make the media free from any
Contaminant.
Methods of sterilization
A. Autoclaving –
•The majority of culture media are sterilized by being
autoclaved.
•This ensures the destruction of bacterial endospores as well as
vegetative cells.
•It is important to sterilize a medium at the correct temperature
and for the correct length of time as instructed in the method
of preparation.
•Under sterilization and over sterilization will affect the quality
of the culture media .
•NB. Most culture media are sterilized at a pressure of 15 lb/in
2
and temperature of
121
o
C for 15 minutes.
31

32. B. Steaming at 100
o
C
•It is used to sterilize media which contain ingredients that
would be inactivated at temperature over 100
o
C.
E.g. Cary-Blair transport media
•Steaming can be performed by placing media in boiling water
for certain time.
32

33. C. Filtration
•This provides a means of removing bacteria from fluids. It is
used mainly to sterilize additives that are heat sensitive and
cannot be autoclaved or heated at 100
o
C.
Eg. Serum and other solutions containing urea and
certain carbohydrates.
–Several different types of filters can be used including those made
from sintered glass or inert cellulose esters (membrane filters).
–They are available in a variety of pore sizes, and 0.22-0.45 µm pore
size is required for sterile filtration.
33

34. Sterility testing
•This step actually comes after the media is dispensed and
ready for use
•For sterile media in screw –cap tubes, bottles, or petridishes
the simplest way to test for contamination is to incubate the
entire batch or 3 –5% of the batch at 35 –37
o
C overnight.
•Contamination by micro organism capable of over night
growth will be shown by turbidity in a fluid medium and
growth on or in a solid medium.
34

35. 3.Addition of heat sensitive ingredients
•Refrigerated heat sensitive ingredients should be first warmed
at room temperature before added to a molten agar medium.
•Using an aseptic technique, the ingredient should be added
when the medium has cooled to 45
o
C and should be
distributed immediately unless further heating is required.
•Heat sensitive ingredients include serum, whole blood, egg
yolk etc.
35

36. 4. pH testing
•The pH of most culture media is near neutral. An exception is
alkaline peptone water.
•The simplest way of testing the pHof a culture medium is to
use narrow range papers or a pH meter.
•A fluid medium can be tested by dipping a narrow range pH
paper in to a sample of the medium when it is at room
temperature and comparing the colour of the paper against the
pH colour chart.
36

37. •An agar medium can be tested by pouring a sample of the
molten medium in to a small beaker or Petri dish and when it
has solidified, laying a narrow range pH paper on it’s surface.
•The colour of the paper is then compared against the pHcolour
chart
•The pH meter can also be used to measure Ph of a fluid or
molten medium.
NB.
–The Ph of a dehydrated medium should not require adjustment provided it has
been prepared correctly using pure water and clean equipment and it has not been
over heated.
–The Ph of other media can be adjusted using 0.1 molar (0.1N) NaOH and 0.1 M
or (0.1N) Hcl.
37

38. 5. Dispensing of culture media
•Most fluid media, unlike solid media, are first dispensed into
screw capped bottles or tubes, and then sterilized by
autoclaving.
•Media should be dispensed in a clean draught-free room using
aseptic technique and sterile container.
38

39. Sterilizing glass Petri dishes, tubes and other glass wares
•Culture media should be dispensed in sterile containers and
glass wares.
•Containers and glass wares are sterilized using dry heat (in dry
heat oven) and a temperature of 160
o
C held for 45 –60
minutes for effective sterilization.
NB: A cooling time is also necessary to enable the items in the oven to cool slowly.
The oven door must not be opened until the temperature inside the oven has fallen
to below 50
o
C. This will avoid cracking the glass ware.
39

40. Dispensing agar media in petridish
1.Lay out the sterile Petri dishes on a level surface
2.Mix the medium gently by rotating the flask or bottle. Avoid forming air
bubbles.
3.Flam sterilize the neck of the flask or bottle and pour 12 –15 ml of
medium into each dish (90 –100 mm diameter)
If air bubbles enter while pouring, rapidly flame the surface of the medium
before gelling occurs. Rotate the dish on the surface of the bench to ensure
an even layer of agar.
4.When the medium has gelled and cooled, stack the plates & tubes and seal
them in plastic bags to prevent loss of moisture and reduce the risk of
contamination.
5.Store at 2 –8
o
C
40

41. Dispensing agar in culture tubes or bottles
•Sterilized agar media are dispensed aseptically half filled in a
sterile screw cup bottles or tubes and allowed to set in a slope
or slant position to increase the surface area for aerobic
inoculation.
Figure. Agar media are dispensed aseptically half filled in a sterile
screw cup bottles or tubes and allowed to set in a slope or slant
position.
41

42. 6. Quality control or performance test
•Performance test is used to check the quality of the culture media
prepared (whether it can be used to perform the intended test) using
control organisms.
•Whenever possible, the regional microbiology laboratory should
supply district laboratories with standardized tested media and when
this is not possible each district laboratory should set up its own
quality control of the media it prepared.
•A set of control organisms (stable stock strains) will need to be
obtained from the regional or central public health laboratory and
the viability of these organisms should be maintained with regular
sub culturing.
42

43. Control of nutrient agar, blood agar, chocolate agar
•Use appropriate control species. Inoculate slopes or quarter plates of the
medium to be tested with a 5 hour broth culture of each control organisms.
•Depending on the species, incubate aerobically or in a carbon dioxide
enriched atmosphere at 35 –37
o
C.
•After over night incubation, examine the cultures for the degree of growth,
size of colonies, and other characteristics such as alpha or beta hemolysis.
•Record the result of each control species and compare with the standard.
43

44. Control of a transport medium
•Immerse in the medium a swab of the specimen containing the pathogen (s)
to be preserved (E.g Urogential swab containing N. gonorrhea in Amies
transport medium)
•Leave the inoculated transport medium at room temperature for the length
of time the medium is intended to preserve the viability of the pathogens it
contains.
•After this time, inoculate the swab on an appropriate medium to check for
viability of the pathogen.
NB. Similar control test is applied for selective, biochemical testing media
and other media
44

45. 5. Storage of culture media
•Plates of culture media should be stored at 2 –8
o
C, preferably
in sealed plastic bags to prevent loss of moisture.
•Antimicrobials in solution form should be stored at –20
O
C.
•All types of prepared media should be stored in the dark.
When in use, the media must be protected from direct light,
especially sun light.
•All culture media must be clearly labeled. Each batch of
prepared medium should be given number and its preparation
date recorded.
45

46. CULTURING OF BACTERIA
Materials for Culturing Bacteria
Basic materials used for culturing bacteria.
•Culture media
•Petri dishes
•Test tubes
•Inoculating loops, straight wire loops
•Bunsen burner
•Incubator
46

47. Depending on the characteristics and nutritional requirement,
bacteria can be inoculated on different culture media hence the
choice of culture media for inoculation of samples depends on:
1.The major pathogens to be isolated, their growth requirements,
and the features by which they are recognized.
2. Whether the specimens being cultured are from sterile sites or
from having normal microbial flora.
NB: Although a selective medium is usually more expensive than a non
selective one, it often avoids sub culturing , isolates a pathogen more
quickly, and makes it easier to differentiate and interpret bacterial growth.
3. Cost, availability, and stability of different media in tropical
countries.
4. Training and experience of laboratory staff in preparing, using,
and quality controlling culture media.
47

48. Inoculation of culture media
Inoculation: -is artificial seeding or introduction of micro
organisms on/in to culture media or animal body.
–Immediately before inoculating a culture medium, check the medium
for visual contamination or any change in its appearance medium for
visual contamination or any change in its appearance which may
indicate deterioration of the medium.
E.g. Darkening of the medium
Any growth of micro organisms
•When inoculating culture media, an aseptic technique must be
used to prevent contamination of specimens and culture media,
and laboratory worker and the environment
48

49. Aseptic technique
•Decontaminate the workbench before and after the work of the day.
•Use facemask and gloves during handling highly infectious specimens.
•Flame sterilize wire loops, straight wires, and metal forceps before and
after use
•Make slide preparation from specimens after inoculating the culture media
NBPrevent aerosol formation as much as possible
•Flame the necks of specimen bottles, culture bottles and tubes after
removing and before replacing caps or plugs.
•When inoculating, do not let the tops or caps of bottles and tubes touch an
unsterile surface. This can be avoided by holding the top cap between
fingers in hands.
•Use a safety cabinet when working with hazardous pathogens and wear
protective clothing.
49

50. Inoculation of Media in Petri dishes
The aim of inoculating media (solid) is to get isolated single
colonies which are derivatives of single bacterium. (i.e. pure
colony).
Up on inoculating the sample successively its distribution will
become decreased and at last end up in isolated colonies.
The technique used to inoculate media in Petri dishes (plate)
must provide single colonies for identification
A pathogen must be isolated in pure culture before it can be
identified and tested for antimicrobial sensitivity.
50

51. •The inoculation of media in Petri dishes is referred to as
‘plating out’ or ‘looping out’.
•It is not necessary to use whole plates of media for every
specimen. Considerable savings can be made by using half or
even a third of a plate, but the area of medium used must be
sufficient to give separate colonies.
•Before inoculating a plate of culture medium, the surface of
the medium must be dried, other wise single colonies will not
be formed.
51

52. Inoculating Techniques
NB: For sputum and stool samples since they have large
number of bacteria, prior to inoculation we have to dilute or
homogenize the samples.
1. Using a sterile loop or swab of the specimen, apply the
inoculation to a small area of the plate.
2. Flame sterilize the loop, when cool or using a second sterile
loop, spread the inoculation systematically. This will ensure
single colony growth
52

53. Figure. Inoculation techniques.
53

54. 54

55. Inoculation of Slopes
•To inoculate slopes (such as Dorset egg medium or TSI) use a
sterile straight wire to streak the inoculation down the center
of the slope and then spread the inoculation in a zigzag pattern
as shown in the figure.
Figure. Inoculation of Slopes.
55

56. •To inoculate a slope and butt medium such as kligler iron agar
(KIA), use a sterile straight wire to stab in to the butt first and
then use the same wire to inoculate the slop in a zigzag pattern.
Inoculation of Stab Media (deeps)
•Use a sterile straight wire to inoculate a stab medium. Stab
through the centre of the medium taking care to with draw the
wire along the line of inoculation with out making further stab
lines.
Fig. Inoculation of a deep (stab)
56

57. Inoculation of fluid media
•Broths and other fluid media are inoculated using a sterile wire
loop, straight wire, or pasture pipette depending on whether
the inoculums is a colony, a fluid culture or a specimen.
•When using a wire loop, hold the bottle or tube at an angel and
rub the loop against the side of the container below the level of
the fluid.
Figure. … Inoculation of fluid media
57

58. Labeling of inoculated media
•Using grease pencil or marker, label inoculated media with the
date and the patient’s number
•always label the base of the culture plate not lids.
Incubation of inoculated media
•Inoculated media should be incubated as soon as possible. A
delay in incubation can affect the viability of pathogens.
•Micro organisms require incubation at the temperature,
humidity & gaseous atmosphere most suited for their
metabolism.
58

59. Temperature of Incubation
•The temperature at which micro-organisms grow best is
referred to as its optimum temperature.
•The temperature selected for routine culturing is 35 -37
o
C,
however, most microbiologists recommend 35
o
C.
•Temperature of growth is also used in the differentiation of
mycobacterium species.
E.g. No growth is produced by M. tuberculosis and H. ulcerans at 25
o
C
where as many opportunistic and saprophytic mycobacteria grow at this
lower temperature.
59

60. •Some exception is that Yersinia enterocolitica grows best at 20
-28
o
C which helps to identify the species.
•Humidity -growth atmosphere which is too dry can affect the
growth and viability of many pathogens.
60

61. Culturing of Anaerobes
•An anaerobic atmosphere is essential for the growth of strict
an aerobes such as clostridium species, bacteroides, and
anaerobic streptococci.
•Anaerobic incubation also helps to differentiate pathogens and
isolate facultative anaerobes from specimen containing
commensals.
Example-Streptococcus pyogens from throat swabs
•The hemolytic reactions of beta -hemolytic streptococci are
also more pronounced following anaerobic incubation.
•To create anaerobic environment a tightly closed container or
jar is required it is usually called Anaerobic jar.
61

62. •
Figure. Anaerobic jar
•There are several techniques for obtaining anaerobic
conditions
1. Commercially produced sachets contain oxygen removing
chemicals
E.g. -Merck anaerocult anaerobic system
62

63. •The sachets contain a mixture of iron powder, citric acid and
sodium carbonate. Addition of a small volume of water
activates the chemical and oxygen is rapidly removed leaving
an aerobic conditions in the anaerobic jar. Some CO
2 is also
produced.
•Palladium pellets are used as catalyst
Figure: Reactions in anaerobic jar
63

64. •Control of anaerocult system –is using anaerobic indicator
strips which will change colour in the absence of oxygen
(anaerobic condition).
2. Copper coated steel wool to remove oxygen
Steel wool + acidified copper sulphate solution –this will coat
the iron and rapidly absorb oxygen when put in a sealed plastic
bag.
3. Use of reducing agents in culture media
E.g. Thioglycollate broth which is used mainly to culture
anaerobes in blood , contains the reducing agent sodium
theioglycolate and the indicator methlene blue to show that the
medium is reduce.
64

65. Culturing in carbon Dioxide
•Carbon dioxide enriched atmosphere is used for the growth of
N. gonorrhea,N. Maningitidis, Brucella species, and
streptococcus pneumonia.
•The simplest and cheapest way of providing a carbon dioxide
enriched atmosphere is to enclose the inoculated plates in an
air tight jar with a lighted candle. As the candle burns, the
oxygen content is reduced releasing a carbon dioxide content
of 3-5% by the time the candle extinguished.
Figure.Culturing in carbon Dioxide
65

66. Appearance of Bacterial colonies on solid Media
Bacterial colonies should be examined in a good light and a low
power magnifying lens can help to see morphological details.
1. When viewed form above: colonies may appear round,
irregular crenated, or branching.
•They may be transparent or opaque and their surface may be
smooth or rough, dull or shiny. Eg. The colonies of
pneumococci have a ringed appearance.
66

67. 2. When viewed form the side: Colonies may appear flat or raised
in varying degrees some times with beveled edges or with a
central elevation or depression.
67

68. 3. When touched with wire loop: Some colonies are soft and
easily emulsified such as S.aureus. Where as others are
difficult to break up such as S. pyogens.
4. The colour of colonies: this also helps to identify bacteria,
especially when using differential media containing indicators.
E.g. -V. cholera in TCBS agar appear Yellow
-Corny bacterium diphteriae in Tellurite agar appear black.
Figure:TCBS Agar -Vibrio chol.
68

69. Changes which may occur in the medium when bacteria are
cultured on solid agar.
•These include hemolytic reactions, pigment production, color
changes surrounding carbohydrate fermenting colonies, and
blackening due to hydrogen sulphide production.
* An example of pigment forming organism is pseudomonas
aeroginosawhich produces a yellow –green color in media
such as blood agar and MacConkey agar.
* An examples of organism that produces a color change is
Vibriochorerawhich is sucrose fermenting, giving a yellow
color in TCBS agar.
69

70. •Blacking due to hydrogen sulphide production is seen with
many Salmonellacultured in kligler iron agar(KIA).
•Hemolytic reaction in blood agar is seen with beta hemolytic
Streptococci (complete hemolysis) and alpha hemolytic
pneumococci(partial hemolysis)
Figure: Beta hemolysis Figure: Alpha hemolysis
NB. Morphological appearance of colonies on blood agar can
vary depending on the species of blood used. E.g. Horse,
sheep, or goat blood.
70

71. Reporting culture results
The manner of reporting culture depends on whether the
specimen is either from a sterile site or from site with a normal
microbial flora.
Sites normally Sterile: Identify and report all bacteria isolated
up to their genera. And if helpful identify the actual species
using bio chemical tests. Sterile sites include blood, bone
marrow, CSF, pleural and peritoneal fluids.
NB. For isolating organism from sterile sites use culture
media which is non selective, and enriched.
71

72. Sites Having a Microbial Flora:
Interpretation requires patients’ clinical data to judge whether
an isolate is a pathogen which causes the patients illness.
The laboratory report should indicate those organisms for
which isolation technique has been performed.
For example when the pathogen have been isolated from
feacal specimen cultured on selective medium like SSA
(salmonella shigela agar). The report should state as ‘no
salmonella or shigella organism isolated’.
72

73. •Sites with normal flora include faeces (stool), sputum, skin,
throat and nose swabs, vagainal, cervical, and urethral swabs.
NB: For isolating organism from sites with normal flora selective
media are much better than general media.
73

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74