Experiment 4

Cultural research method

 Outline :
1. Study and differentiate between different cultural types (pure and mix).
2. Study and differentiate between different microbiological media.
3. Streaking technique to make pure cultures (using 3-loop method).

 Introduction :

- A culture can be defined as the division of an organism “in vitro” through the
presence of special conditions such as: nutritional media, pH, temperature,….etc.

*Note*
Other factors should be taken into consideration depend on the type of bacteria and the
purpose for which they are cultivated

- In medical microbiology , the work is done continuously according to the following

scheme:

1. Cultivation of samples like: pus ,abscess ,urine ,CSF , blood …..etc. theses
samples contain the infectious agents and should be cultivated on different media for
the diagnosis like Chocolate agar, Blood agar, Nutrient agar, MacConkey agar and in a
tube containing meat extract for enrichment of the sample.
2. Identification of the microorganism grown on the media
 3. Decision making about the pathogenic bacteria causing the infection.
4. Determine the sensitivity against different chemotherapeutic agents prescribed for
the infectious agents.
- The samples are cultured either on solid medium or semi-solid or in liquid medium.
The choice of the media taken depends on the suggested previous clinical diagnosis
(normally according to the above mentioned media, or according to media suggested
by the American Society for Microbiology depending on the microorganism expected).

- Primary culture: is the culture of the first sample cultivated

- According to the first sample materials cultivated, the primary culture will show
either :

a. Mixed culture: Contains more than one type of bacteria (especially if taken from non-
sterile body sites as from skin injury or wound or GIT, or mouth, nasal, throat, stool,
…etc).
or it could be more than one pathogen causing the disease (double infection as for
example in urine infections, or in injuries, or necrotizing fasciitis, …etc ). Now, once
we know the infectious agent, we take it with the loop and sub cultivate on culture
media alone (This is then called Pure or monoculture.

b. Pure or Mono culture: Contains one type of bacteria, either it arises from samples
taken from sterile parts of the body ex. CSF and blood, etc or it arises after
purification from mixed culture (It becomes a pure culture and at the same time
is considered as secondary culture since it is taken from primary culture).

- Secondary culture (subculture): Subculture from the primary culture

- The Colony is cluster of organisms growing on the surface or within a solid medium,
arose from single original cell, well isolated, recognized, originated from one or more
cells and contain genetically identical organisms that have almost 107 cells.

- Normal microbial flora: (Commensals) microorganisms that are frequently found in

particular anatomic sites of healthy individuals. Healthy adults have 10 trillion cells and
routinely harbors 100 trillion microbes.
 Additional attachment for culture bacteria:
https://youtu.be/949qQtAnjIc

Types of sampling test:

1- Invasive sample
2- Non-invasive sample
3-
The major difference between invasive and non-invasive samples :
1. Invasive sample: Are samples taken from sterile body sites (No bacteria available
normally). If these sites are infected, then the infection is normally due to one single
bacterium, which could invade the body site and go to these sterile body sites, then it
will be as pure culture after primary cultivation. Examples CSF sample, Blood sample,
sample from tissue inside the body
2. non-invasive samples are samples taken from sites with normal flora available on the
site of infection, here the primary culture contains mostly mixed bacteria. Examples are
samples taken from skin infections, wounds, mouth, nose, nasopharyngeal, urethra,
vaginal, gastrointestinal, stool, sputum, upper respiratory tract.

Microbiological Culture Media

The survival or growth of microorganisms depends on available nutrients and a favorable
growth environment. In the laboratory, the nutrient preparations that are used for culturing
microorganisms are called media (singular, medium).

Culture media can be classified on the basis of several parameters: the chemical
constituents from which they are made, their physical nature, and their function.

Based on Physical nature; there are several different forms of media, which include
liquid or broth medium, semisolid and solid agars. The major difference among these
media is that solid and semisolid media contain a solidifying agent (usually agar),
whereas a liquid medium does not.

Note: The most common solidifying agent is agar, which is found in many marine
plants and is usually extracted from red algae.
 Based on Chemical composition, a medium in which all chemical components are
known is defined or synthetic medium, whereas media that contains some ingredients
of unknown chemical composition are complex media i.e. MacConky agar.
Complex media contain undefined components like peptone, meat extract, and yeast
extract.

Based on the functional types of media, media is classified into the following types:

a. General purposes media (supportive): contains the simplest nutritional requirements

needed to support the growth of many microorganisms that don’t need special
nutritional requirements (e.g., Nutrient agar or nutrient broth)

Figure (1): Nutrient agar and nutrient broth media

b. Enriched media: they are specially fortified media containing special nutrients like
whole or lysed blood, serum, and special extracts or nutrients to encourage the growth
of fastidious microbes (e.g., Blood agar and chocolate agar, or also meat extract broth).
 Figure (2): blood agar, chocolate and and meat extract broth .

c. Selective media: Broth medium for optimal and fast growth (e.g., Sodium
Selenite broth). This media is made for the isolation of salmonella species, therefore is
considered as selective not enrichment media

Figure (3): Selenite broth

d. Selective media: medium this enhances the growth of a group of bacteria (mostly
pathogen) and inhibits the growth of others that are either normal flora or
contamination (e.g., MacConky agar and eosin Methylene blue agar, or also selenite
broth or selenite agar,…and many others).

Figure (4): Eosin Methylene blue agar

e. Special media: media used for special types of microorganisms such as Gonococci,
Candida, Mycoplasma, Fungi, etc…These media contains inhibitory substances for
inhibition of other bacteria and keeping the required bacteria.

f. Differential media: are media that distinguish among different groups of microbes
and even permit tentative identification of microorganisms based on their biological
characteristics.

Blood Agar
Blood Agar is a highly nutritious, is an enriched medium for the isolation and cultivation
of non-fastidious and fastidious microorganism. Also, it is a differential media, in which
hemolytic and non-hemolytic microorganisms can be easily distinguished by examining
for zones of blood lysis. The hemolysis Patterns on Blood Agar includes the following:
 Figure (5): The hemolysis Patterns on Blood

Beta hemolysis (- hemolysis) means that the bacteria's hemolytic enzymes
completely break down the blood cells, resulting in clear hemolytic zones around
bacterial colonies.

Alpha hemolysis (α-hemolysis) means that the bacterial enzymes only partially break
down the blood cells. This results in the media showing a yellowish/greenish/brownish
discoloration around the colony, indicating incomplete hemolysis.

Gamma hemolysis (γ) is essentially no hemolysis at all; that the bacteria have no
effect on the red blood cells and there is no change of the color of the medium.

MacConkey agar
MacConkey agar is a selective agar, where only gram negative can grow on it. It
contains inhibitory materials for gram-positive bacteria. These materials are crystal
violet and bile salts, which inhibits gram-positive bacteria, therefore only gram-
negative bacteria can grow.

MacConky agar is considered as selective and differential media. Selective because

they let only the gram negative bacteria to grow, and differential because we can
differentiate between lactose fermenters (red colonies) and lactose non-fermenters (pale
or transparent colonies). Eosin Methylene blue (EMB) agar is another example.
 Figure (6): MacConkey agar

In addition, MacConkey agar is selective for Gram-negative bacteria because of the

presence of bile salts and crystal violet that inhibit the growth of gram-positive bacteria.
Also, MacConkey agar differentiate between them by their ability to ferment lactose
sugar by using neutral red indicator (Phenol red) which gives a red color under acidic
pH and yellow color under basic pH; when the bacteria is Lactose fermenter such as
Escherichia coli, Enterobacter and Klebsiella, the bacteria will produce acids, which
lowers the pH of the agar below 6.8 and results in the appearance of red/pink colonies.
Whereas the Lactose non- fermenting bacteria such as Salmonella, Proteus species and
Shigella cannot utilize lactose, and will use peptone instead. This forms ammonia, which
raises the pH of the agar, and leads to the formation of yellow/colorless colonies.
Figure (7): Macconkey’s Agar
Figure (8): Eosin Methylene blue (EMB) agar
Experiment 1: Streaking using a 3 loop method
(1) Flame your loop and allow it to cool on the agar at least 10 to 15 seconds before
streaking the culture. Wait until the loop stops “hissing.”
(2) Use a loopful of culture from either broth or solid medium; streak the first sector as
it shown below in figure (1).
(3) Sterilize the loop and then let it cool.
(4) Rotate the plate 90 degree while carefully keeping in mind where the initial streaks
ended and cross over the streaks in Sector A. Streak out any bacteria picked up from
sector A into sector B.
(5) Sterilize the loop again. Rotate the plate and cross over the streaks in Sector B.
Streak out any bacteria picked up from Sector B into Sector C.
(6) Cross over the streaks in sector C into sector D without further sterilization.
(Sector C and sector D are streaked using the same loop)
(7) Sterilize the loop when finishing your streaking.
(8) An inoculated plate is always incubated in an inverted position to prevent
condensation from falling onto the surface of the plate and interfering with discrete
colony formation.
(Incubate for 24 hours at 37 C).
(9) Study the colony morphology (description of the bacterial colonies) of the bacteria
that you’ve cultured; using the table in the next page.

Additional attachment for streaking using a 3 loop method:

https://youtu.be/eVh_YPNr6lI
Figure (9) : 3 loop method streaking
 How to label your plate:
Petri plates are labeled at the back side of your plate (at the edges) not on the lid.
Label all experimental material with:
a. Your Name
b. Date
c. Exercise number
d. Lab section
e. Specimen
For example: Muhammad/05.04.2022/ section 1/ ex. 3/ Escherichia coli

Experiment 2: Differentiation of bacteria on different agar

media.

 Materials:
o A normal saline suspension in a screw cap tube that contains (Escherichia coli, Proteus
mirabilis, and Staphylococcus aureus) bacteria.
o Inoculating loop
o One plate of Nutrient agar medium.
o One plate of MacConkey agar medium.
o One plate of Blood agar medium.
o Bunsen burner.

 Procedure:
o Each group works on one normal saline suspension that contains (Escherichia coli,
Proteus mirabilis, and Staphylococcus aureus).
o The mentioned above agar media will be cultivated with a loopful of the normal saline
suspension using the 3-loop streaking method, giving a mixed culture.
o Study the colony morphology (description of the bacterial colonies) of the bacteria that
you’ve cultured; using the table in the next page.
o Record your observations and fill the table below:
 Microbiology Lab Report 2
Cultural research method

Student Name: Student ID:

1- Fill in The Table: (6 marks)

Name Of Bacteria

Observation Result

Gram Stain Result

Microscopic
Examination

Type Of Hemolysis On
Blood Agar

MacConkey Agar
(Fermenter or Non
Fermenter )

Form Colony

Colony Elevation

Colony Margin
2- Both blood agar media and MacConkey agar media are differential media
explain…..! (2 marks)

3- Fill in The Table: (2 marks)

Primary culture Number of Bacteria Type Example of Samples

Mixed culture

Pure (Mono) culture