Culture media, and Pure culture method
Culture Tubes and Petri Dishes
We use glass test tubes and glass or plastic Petri dishes to cultivate microorganisms. We can add a
suitable nutrient medium in the form of broth or agar to the tubes, while we use only a solid medium in Petri
dishes. We maintain a sterile environment in culture tubes by various types of closures. Historically, the first
type, a cotton plug, was developed by Heinrich G. F Schröeder and Theodor von Dusch in the nineteenth
century. Today most laboratories use sleeve-like caps (Morton closures) made of metal, such as stainless
steel, or heat-resistant plastics. The advantage of these closures over the cotton plug is that they are labor-
saving and, most of all, that they slip on and off the test tubes easily.
Petri dishes provide a larger surface area for growth and cultivation. They consist of a bottom dish
portion that contains the medium and a larger top portion that serves as a loose cover. Petri dishes are
manufactured in various sizes to meet different experimental requirements. For routine purposes, we use
dishes approximately 15 cm in diameter. The sterile agar medium is dispensed to previously sterilized dishes
from molten agar deep tubes containing 15 ml to 20 ml of medium, or from a molten sterile medium
prepared in bulk and contained in 250-, 500-, and 1000-ml flasks, depending on the volume of medium
required. When cooled to 40°C, the medium will solidify. Remember that after inoculation, Petri dishes are
incubated in an inverted position (top down) to prevent condensation formed on the cover during
solidification from dropping down onto the surface of the hardened agar. For this reason, we should label
Petri dishes on the bottom of the dish. This makes it easier to read the label and minimizes confusion if two
Petri dish covers are interchanged. Figure 3.1 illustrates some of the culture vessels used in the laboratory.
Built-in ridges on tube closures and Petri dishes provide small gaps necessary for the exchange of air.
Test tube rack with tubes showing various closures. Petri dish DeLong shaker flask with closure
A. Bacteriological tube
B. Screw cap
C. Plastic closure Figure 3.1. Culture vessels
D. Metal closure
E. Nonabsorbent cotton 1
Media Classifications and Functions
Media are categorized according to their function and use. In diagnostic bacteriology there are four general
categories of media: basic, enrichment, selective, and differential.
1. General purpose media/ Basic media
Basal media are basically simple media that supports most non-fastidious bacteria. Peptone water, nutrient
broth and nutrient agar are considered as basal medium. These media (e.g Nutrient Agar, Nutrient Broth) are generally
used for the primary isolation of microorganisms.
Nutrient Agar
Nutrient
Broth
2. Enriched medium (Added growth factors)
Addition of extra nutrients in the form of blood, serum, egg yolk etc, to basal medium makes them enriched
media. Enriched media are used to grow nutritionally exacting (fastidious) bacteria. B lood agar, chocolate agar,
Loeffler’s serum slope etc are few of the enriched media. Blood agar is prepared by adding 5-10% (by volume) to a
blood agar base. Chocolate agar is also known as heated blood agar or lysed blood agar.
Blood agar
Chocolate agar
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3. Selective medium
Selective medium is designed to suppress the growth of some microorganisms while allowing the growth of
others.
Mannitol Salt Agar and Salt Milk Agar used to recover S. aureus contain 10% NaCl.
MacConkey’s Agar used for Enterobacteriaceae members contains Bile salt that inhibits most gram positive bacteria.
Mannitol Salt Agar (MSA)
MacConkey’s Agar
4. Differential/ indicator medium
Certain media are designed in such a way that different bacteria can be recognized on the basis of their colony color.
Various approaches include incorporation of dyes, metabolic substrates etc., so that those bacteria that utilize them
appear as differently colored colonies. Such media are called differential media or indicator media. Differential media
allow the growth of more than one microorganism of
interest but with morphologically distinguishable colonies.
Mannitol salts agar (mannitol fermentation = yellow)
MacConkey agar (lactose fermenters, Pink colonies whereas non- lactose fermenter produces pale or colorless
colonies.
MacConkey Agar
A: Enterobacter aerogenes (lactose fermenters)
B: Shigella dysenteriae (non- lactose fermenter)
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Pure culture method (Streak-Plate Method; Isolation of Bacterial Colonies Using the
Quadrant Method)
The streak-plate procedure is designed to isolate pure cultures of bacteria, or colonies, from mixed populations
by simple mechanical separation. Single colonies are comprised of millions of cells growing in a cluster on or within an
agar plate (Figure 3.2). A colony, unlike a single cell, is visible to the naked eye. In theory, all the cells in a colony are
derived from a single bacterium initially deposited on the plate and thus are referred to as a clone, or cluster of
genetically identical cells.
Bacteria exist in a variety of shapes and sizes. For example, individual Escherichia coli cells are rod-shaped
with an average length of 2 μm and width of 0.5 μm while Streptococcus cells are spherical with an average diameter
of 1 μm. Some bacteria (such as E. coli) exist as single cells while others form distinct patterns of association.
Streptococcus, for instance, grow in pairs or form chains or clusters of cells. It is generally assumed that a single colony
arises from a single cell undergoing binary fission; however, this assumption is not true for those bacteria that naturally
exist as pairs, chains, or clusters or that divide by other mechanisms. Alternatively, if too many bacteria are plated, then
overlap of cells may occur and increase the probability of two or more bacteria giving rise to what appears to be a single
colony. To avoid these complications when describing or enumerating bacterial cultures growing on a solid medium,
colonies are referred to as colony forming units (CFU).
With the streak-plate procedure, a mixture of cells is spread over the surface of a semi-solid, agar-based
nutrient medium in a Petri dish such that fewer and fewer bacterial cells are deposited at widely separated points on the
surface of the medium and, following incubation, develop into colonies.
Purpose of streaking: To obtain pure, isolated colonies.
Principle: By spreading a large amount of bacteria over the large surface area of a plate, the amount of bacteria is
diluted until individual cells are spread on the surface of the plate. Each individual cell grows into a single colony. All
of the cells in this colony are genetically identical.
Fig. 3.2 Example of single colonies on a plate.
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Procedure
(A) In a streak plate, a single loopful of a culture is spread four or five times in the first quadrant.
(B) The loop is flamed and allowed to cool for about 10 sec. touching the loop to an uninoculated area of the plate will
ensure that it is cool. Once cool, five or six streaks are made from quadrant 1 into quadrant 2.
(C) Six to seven streaks are made from quadrant 2 into quadrant 3.
(D) After flaming and cooling once more, several streaks are made from quadrant 3, using up all of the uninoculated
space on the plate.
Finally, the loop is flamed before being placed aside.
B
D
C
Streak pattern
Bacterial growth pattern
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Bacterial colonies may be described on the basis of their form, elevation, and margins.
References
1. Connie R. Mahon, Donald C. Lehman, George Manuselis . Textbook of Diagnostic Microbiology, 6th Edition. 2019.
2. Frank E. Berkowitz, Robert C. Jerris. Practical Medical Microbiology for Clinicians. 2016.
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Staining
Microscopic examination is used to determine a cell’s size and shape as well as the often characteristic
arrangement the cells of a given species assume as they multiply. The initial classification of an isolate is
absolutely dependent upon the cells’ shape (Figure 4.1), arrangement (Figure 4.2), and staining characteristics,
although complete identification is not possible from these aspects alone.
Fig. 4.1 Bacteria are organized into four groups according to shape. Coccus (cocci): spherical cells;
Bacillus (bacilli) rod or cylinders; Spiral: usually occur singly and can be subdivided into loose, tight,
and comma shaped and Pleomorphic: shape ranging from cocci to rods.
Fig. 4.2 Bacterial Arrangements: Some occur singly, such as spirilla and most bacilli. Some occur in
pairs (diplococci), some occur in clusters, bunches, or groups, some can be arranged in a palisade or a
“Chinese Letter” pattern.
Bacterial cells are essentially transparent, and a careful examination of them involves the same problems
as a careful examination of ice cubes in a glass of water. Because the cells and their background do not contrast
well, they are difficult to visualize. Colored stains are used to increase the contrast between bacterial cells and their
background, and while these stains differ in many ways, they share a basic chemistry that allows them to interact
with cells.
Staining is 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. Depending on the type of
dye, the positive or the negative ion may be the chromophore (the colored ion); the other, uncolored ion is called
the counter ion. 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 (Table 1).
Dyes are selected for staining based on the chemical properties of the dye and the specimen being
observed, which determine how the dye will interact with the specimen. 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. However, there are scenarios in which it is advantageous to
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use a negative stain, which is absorbed by the background but not by the cells or organisms in the specimen (In
negative staining, the background is stained, leaving clear cells visible against a dark background).
Because cells typically have negatively charged cell walls, the positive chromophores in basic dyes tend to
stick to the cell walls, making them positive stains. Thus, commonly used basic dyes such as basic fuchsin,
crystal violet, malachite green, methylene blue, and safranin typically serve as positive stains. On the other
hand, the negatively charged chromophores in acidic dyes are repelled by negatively charged cell walls, making
them negative stains. Commonly used acidic dyes include India ink, nigrosine, 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. In simple staining, a single dye is used to emphasize particular structures in the specimen. A simple stain
will generally make all of the organisms in a sample appear to be the same color, even if the sample contains more
than one type of organism.
Although simple and negative staining increases the contrast between a cell and its background, making it
easier to determine the size, shape, and arrangement of a particular bacterium, they provide little information
beyond this. One advantage seen with negative staining is a more accurate determination of the size of a bacterial
cell.
In contrast, differential staining distinguishes organisms based on their interactions with multiple stains.
In other words, two organisms in a differentially stained sample may appear to be different colors. Differential
staining techniques commonly used in clinical settings include Gram staining, acid-fast staining, endospore
staining, flagella staining, and capsule staining.
Table 1. Ionizable Dyes
Type of Dye Example Characteristics
Have positively charged group; bind to negatively charged
Methylene blue, basic fuchsin, crystal
Basic dyes molecules such as nucleic acid, many proteins, and the
violet, safranin, malachite green
surfaces of bacterial cells
Eosin, rose bengal, and acidic fuchsin,
In their ionized form, have a negative charge and bind to
Acidic dyes possess groups such as carboxyls (-COOH)
positively charged cell structures
and phenolic hydroxyls (-OH)
GRAM STAIN
The Gram stain is a differential stain which distinguishes bacteria based on cell wall properties. Bacterial
cell walls are composed primarily of peptidoglycan and bacteria can be classified into two main groups dependent
on the amount of peptidoglycan present in their cell wall. Gram-positive organisms have a thick layer of
peptidoglycan, whereas Gram-negative organisms have a thin layer of peptidoglycan, plus an additional outer
membrane that is absent in Gram-positive organisms.
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In the Gram staining procedure, the primary stain is crystal violet, and all cells take up the purple crystal
violet stain. Following the primary stain, Gram’s Iodine is applied to the bacterial smears. The iodine acts as a
mordant, enhancing the ability of the stain to enter and bind to the bacteria. Specifically, the iodine binds with
crystal violet and locks it into peptidoglycan of bacteria. It also intensifies the purple color. The decolorizing agent
used in the gram staining procedure is 95% ethanol, which is a lipid solvent that melts the Gram negative outer
membrane and leads to decolorization of Gram-negative cells. It also dehydrates proteins, helping the primary stain
to remain in Gram-positive cell walls. The counter stain then used is Safranin, which stains the decolorized Gram-
negative cells pink. Thus, at the end of the staining procedure, Gram-positive cells are purple and Gram-negative
cells are pink. Note: It is preferable to use fresh cultures for the Gram stain. Old cultures may stain “Gram-variable”
(a mix of purple and pink) because they decolorize easily.
Smear Preparation
A thin layer of the specimen is spread on the slide called a smear.
1. If working from a solid medium, add one drop (only one drop) of water to your specimen with a water bottle. If
using a broth medium, do not add the water.
2. Prepare a smear of bacteria by aseptically adding a loop of the culture to the center of a slide.
Be sure to flame sterilize your loop every time you enter the culture tube.
3. Spread the culture over the slide, covering an area about the size of a quarter.
4. Allow the smear to air-dry completely. This should take 5–10 min.
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Smear Fixation
The “fixing” of a sample refers to the process of attaching cells to a slide. Fixation is often achieved either
by heating (heat fixing) or chemically treating the specimen. 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.
Heat-fix the smear by passing the slide, with the smear on top, through the flame of your Bunsen burner
three times.
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Procedure for making a bacterial smear
One Loopful
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Simple Stain Procedure
6. Flood the smear with methylene blue for 1 min.
7. Gently wash the smear with water (3–4 sec).
8. Blot the slide with bibulous paper to remove excess water.
9. Examine the slide under oil immersion.
Rinsing off a slide.
Gram Staining Procedure:
1. Prepare a heat-fixed smear of sample bacteria.
2. Allow the smear to air-dry completely. This should take 5–10 min.
3. Heat-fix the smear by passing the slide, with the smear on top, through the flame of your Bunsen burner three times.
4. Place the slide on a staining tray, and cover the smear with crystal violet. Allow to stain for 1 min.
3. Tilt the slide and gently rinse with distilled water until the stain is removed.
4. Cover the smear with Gram’s Iodine for 1 min.
5. Tilt the slide and gently rinse with distilled water.
6. IMPORTANT STEP: Tilt the slide and let 2-3 drops of Decolorizer run over the slide.
If the last drop is still purple, continue decolorizing, 2-3 drops at a time, until the decolorizer runs clear. (This should take 5–15
sec in most cases.) Rinse with distilled water.
7. Cover the smear with Safranin, and stain for 1 min.
8. Tilt the slide and rinse with distilled water.
9. Blot the slide with bibulous paper to remove excess water.
9. Observe the slide under oil immersion. The Gram staining procedure.
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Crystal violet - stains both Gram negative and positive bacteria
Gram's iodine - fixes the stain in Gram positive bacteria
Ethanol or acetone - washes the stain from Gram negative bacteria
Safranin - counterstain, will re-stain Gram negative bacteria while
not interfering with the previous stain in gram positive bacteria
References
1. Connie R. Mahon, Donald C. Lehman, George Manuselis . Textbook of Diagnostic Microbiology, 6th Edition. 2019.
2. Frank E. Berkowitz, Robert C. Jerris. Practical Medical Microbiology for Clinicians. 2016.
3. Alfred E. Brown, Heidi R. Smith. Benson's Microbiological Applications: Laboratory Manual in General Microbiology, Complete Version
14th Edition. 2017.