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Blood Agar Definition, Preparation, Composition, Application, and limitation.

production of blood agar after the addition of liquid blood.

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Blood agar provides common nutrients as well as 5% sheep blood. It is beneficial for cultivating finicky organisms and assessing an organism’s hemolytic capabilities.

Some bacteria manufacture exoenzymes called hemolysins that lyse red blood cells and destroy haemoglobin. Various forms of hemolysins can be produced by bacteria. Beta-hemolysin totally degrades red blood cells and haemoglobin. This results in a clean area surrounding the bacterial development. These effects are known as -hemolysis (beta hemolysis). Alpha-hemolysin partially degrades red blood cells, leaving behind a greenish hue. This process is known as -hemolysis (alpha hemolysis). The greenish hue results from the presence of biliverdin, which is a byproduct of haemoglobin breakdown. If the organism does not create hemolysins or break down blood cells, there will be no clearance. This is known as -hemolysis (gamma hemolysis).

Streptococci-produced hemolysins operate better in an anaerobic environment. As a result, it is routine procedure to streak a blood plate and then pierce the loop into the agar to create an area with a reduced oxygen content where streptolysins may break down blood cells more effectively.

This streptococcus pyogenes exhibits β-hemolysis.
This streptococcus pyogenes exhibits β-hemolysis.

History

Uncertain is the history of blood agar as it exists today. Blood may have been used as a nutritional addition in culture media prior to the invention of agar. Muir and Ritchie acknowledge its inclusion in their 1903 Manual of Bacteriology before discussing “agar-agar” as a replacement for gelatin as a solidifying agent. In the same discussion, however, they remark that Robert Koch preferred plates poured with a mixture of bacterial inocula and molten gelatin to plates with material streaked across the top. Koch advised “strong and, whenever feasible, transparent” media.

It appears that pour plates were the standard method for many years due to surface contamination issues during incubation. (It should be remembered that agar “plates” were originally sterilised flat glass plates, not Petrie dishes as they are known now.) A method for adding blood to agar media is described in Bulloch’s 1938 paper. The Development of Bacteriology: Blood from humans or animals may be used. Utilize “sloped tubes” containing agar… First cleanse a finger with 1-1000 corrosive sublimate, allow it to dry, and then remove the sublimate with absolute alcohol. Permit the ethanol to evaporate. Prick with a needle sterilised by heat, then collect a drop of blood in the loop of a sterile platinum wire. The extra blood drains away, leaving a film on the surface.

To ensure that the tubes are sterile, cover them with India-rubber caps and incubate them for one to two days at 37°C before to use. Agar spread in a thin layer in a Petri plate may be smeared with blood and utilised for culture in the same manner. It seems prudent to use the blood of the investigated race for examining diseases affecting races other than whites. Anyone interested in the history of microbiology should read Wolfgang Hesse’s biography of his ancestors, Walther and Angelina (“Lina”) (below). Koch was Walther’s mentor, and Angelina was his assistant and illustrator.

Prior to the end of 1882, Walther was frustrated by the summertime melting of his gelatin-coated culture tubes. He questioned Lina about her puddings and jellies, which remained solid despite the warm temperatures. It would appear that she learned about agar from a former neighbour (who had emigrated from Java where agar was a common food additive).

Although there is no recorded evidence, it is plausible that Walther added blood to his cooled, melted agar in the same manner as Lina did with her fruit and meat liquids!

What is Blood Agar?

Blood Agar is an enriched medium provided with multiple nutrients that generally comes as a basal media for the preparation of blood agar by supplementation with blood.

  • Blood Agar comes as a base medium that is able to be used for its role as an all-purpose growth medium. The medium used for the base can be Columbia Agar or Tryptic Soy Agar.
  • The blood agar medium is a fantastic medium to cultivate extremely discerning bacteria that require specific nutrients, and do not grow rapidly on other media such as Agar for Nutrients. Agar.
  • Around 5% of blood of mammals that have been defibrinated (human sheep, horses) will be added autoclaved basal medium to make blood medium for agar.
  • It is an enhanced medium that aids in the development of fastidious bacteria. It also reduces the growth of bacteria, such as Neisseria as well as Haemophilus..
  • To promote the development of these bacteria, the inhibitors present in blood must be eliminated by heating blood Agar. The heated blood agar transforms into Chocolate Agar which supports Neisseria and Haemophilus.
  • One of the primary applications of blood agar is to study the hemolysis triggered by the growth of bacteria, that can later be used to determine the identity of the organism.
  • Blood agar is mainly used to cultivate pathogenic organisms capable of creating extracellular enzymes that can cause hemolysis in blood.
  • We can make the blood agar specific for specific pathogens through the addition of chemicals, antibiotics or dyes. Examples include crystal violet blood agar that is used to identify Streptococcus Pyogens from swabs of the throat and neomycin and kanamycin blood agar that is used to identify anaerobes in pus.
  • Within the US “blood agar” is generally prepared using Tryptic Soy Agar as well as Columbia Agar base that contains 5% sheep blood. 
  • Horse blood or rabbit blood can be used to develop NAD-dependent organisms like as Haemophilus species, however, the hemolytic patterns could be different from those in sheep blood. (Human blood is generally not recommended due to the possibility of being exposed to blood-borne pathogens , including HIV or Hepatitis.)

What is this medium used for?

Some bacteria are more demanding in the nutrients they require for growth, a feature that leads in their being labelled fastidious (or “picky”) (or “picky”). One technique to encourage the growth of medically-important fastidious bacteria is to cultivate them on a medium containing defibrinated blood (blood with clotting proteins removed) (blood with clotting proteins removed). Blood agar is a bright crimson, opaque medium. The range of complex nutrients contained in blood enables the growth of most bacteria, fastidious and otherwise, that would be encountered by students. Such media are said to be complicated (incapable of being chemically replicated) and enriched (having an uncommonly rich array of nutrients) (containing an uncommonly rich array of nutrients).

In addition to its role in encouraging the growth of difficult-to-culture organisms, blood agar has a differential function. Bacteria growing on blood agar can be classed in part on what they do to the red blood cells integrated into the media. Some bacteria create hemolysins, enzymes that damage red blood cells (hemo = blood, lysin = to split). Hemolysins can kill the cells and release the haemoglobin into the media. As the haemoglobin is exposed to the chemicals in the agar, its typical red hue is altered. This kind of hemolysis, alpha-hemolysis, turns the media under the bacterial growth brown-green. Other bacteria are capable of digesting the haemoglobin produced as they kill red blood cells. The upshot of this complete hemolysis, dubbed beta-hemolysis, is clearance of the media under the bacterial colonies. The medium is transformed from opaque to transparent. Other bacteria leave red blood cells essentially intact. The medium is not stained or cleared by growth. Such bacteria are said to be gamma-hemolytic.

Blood Agar Composition 

Like many other nutritional media, Blood Agar includes at least one protein source salt, as well as beef extract to provide minerals and vitamins. In addition to these ingredients, 5percent defibrinated mammalian red blood is added to the medium. The blood agar base can be available for sale by various sellers and can be made in the laboratory when the ingredients readily available. Its exact formula for blood agar is described below:

IngredientsGram/liter
Peptone10.0
Tryptose10.0
Sodium chloride5.0
Agar15.0

In addition to the base medium, 5 % sterile mammalian blood is added after autoclaving prior to pouring on the plates.

Choice of the Blood

Blood agar plates are best prepared with sheep blood, followed by horse, rabbit, or goat blood.

Human blood, especially citrated donor blood that has expired, should not be utilised because it may contain chemicals that hinder the growth of some pathogens. Residual antibiotics in the host’s blood as well as antibodies such as ASO or anti-M protein may inhibit the growth of S. pyogenes. Citrate inhibits beta-hemolytic streptococci growth. Human blood that is infected may also include infectious pathogens.

Principle of Blood Agar

  • If blood is added, blood agar becomes an enriched nutrient media that can be used to cultivate fastidious organisms; yet, it can also be used as a general medium in its absence.
  • When blood is given to a nutrient-poor media, it gives the fastidious microbes with the extra growth elements they need to thrive.
  • Seeing how various bacteria react to hemolysis in the blood is also a useful tool. But the hemolytic reactions are species-specific.
  • Sheep blood is widely used because it is effective against Group A Streptococci, but it cannot foster the development of Haemophilus haemolyticus. As a result of a lack of pyridine nucleotides in sheep’s blood, this is the case.
  • Horse blood is ideal for H. haemolyticus growth and hemolysis, and the resulting hemolysis pattern may resemble that caused by Streptococcus pyogenes when grown on sheep blood.
  • Peptone and tryptose supplement the blood in the medium by supplying the bacteria with carbon, nitrogen, amino acids, vitamins, and minerals. Water solubility of peptone and tryptose facilitates its uptake by the organism.
  • To prevent the medium’s pH from fluctuating too much while the organisms are growing, sodium chloride is added to keep the osmotic balance constant.
  • The nutrients can be more readily absorbed by the bacteria since they can dissolve in the distilled water.
  • In order to observe colony shape and count the organism, agar is used as a hardening agent to give a stable surface for the organism to grow on.
  • To identify phosphate-producing Staphylococci, phenolphthalein phosphate is sometimes added to the medium, along with salt and agar for measuring surface contamination.

Blood Agar Hemolysis/Greening reaction on blood agar

Hemolysis is the process of destroying red blood cells within the blood because of extracellular enzymes that are produced by specific bacteria. The extracellular enzymes made from these bacteria can be referred to as hemolysins, which radiate outwards from colonies, leading to complete and partial destruction of red blood cells. Different types of hemolysis are visible on blood agar, which can be distinguished from an area of hemolysis within the colonies that are growing.

Four types of hemolysis are produced in sheep blood agar namely; alpha (α) hemolysis, beta (β) hemolysis, gamma (γ) hemolysis, and alpha prime or wide zone alpha hemolysis.

Blood Agar and Hemolysis
Blood agar plate results – Blood Agar and Hemolysis

a. Alpha hemolysis on Blood Agar

  • Alpha hemolysis (α) is the transformation of red blood cell haemoglobin into methemoglobin in the surrounding medium. This causes the medium to become green or brown in colour.
  • The colour is analogous to “bruising” the cells. The cell membrane is intact upon microscopic examination of alpha-hemolyzed red blood cells, indicating that it is not, in fact, lysis.
  • Some authors of textbooks refer to alpha as “partial hemolysis,” which might be confusing to students. Importantly, this “partial” or “incomplete” hemolysis should not be confused with the “weak” or “subtle” lysis of Streptococcus agalactiae or Listeria monocytogenes, as described above.
  • Never will beta hemolysis result in brown or green staining of the surrounding medium’s cells. Many alpha hemolytic organisms will become clearer after prolonged incubation, but if the surrounding media has any brown or green hues, the “hemolysis” is still labelled “alpha.”
Alpha hemolysis on Blood Agar
Alpha hemolysis on Blood Agar – (A) Alpha-hemolytic Streptococcus species “Viridans group” streptococci, including species such as the Streptococcus mutans, mitis, and salivarius groups display alpha hemolysis. (B) Alpha hemolysis of Streptococcus pneumoniae (Encapsulated strain).

b. Beta hemolysis on Blood Agar

Complete or genuine lysis of red blood cells characterises beta hemolysis (β). The colony is surrounded by a clean zone that approaches the colour and transparency of the base medium. Numerous bacterial species generate toxins that are capable of damaging red blood cells.

Beta hemolysis on Blood Agar
Beta hemolysis on Blood Agar – (A) Beta hemolytic Streptococcus species, Streptococcus pyogenes (transmitted light) (Lancefield group A). (B) Normal Upper respiratory flora mixed with betahemolytic Streptococcus species. (The presence of beta-hemolytic colonies indicates the possibility of Streptococcus pyogenesinfection.) (C) Same blood agar plate as Figure 2 demonstrating that the beta hemolysis of Streptococcus pyogenes is so complete that print my be read through the resulting transparent medium.

Certain species create several poisons or exhibit variable degrees of beta hemolysis.

  • One example is the rare Streptococcus pyogenes strain that produces solely an oxygen-labile hemolysin (“Streptolysin O”). In other words, hemolysin is only active when oxygen levels are low. A pour plate, agar overlay, or anaerobic incubation can be used to demonstrate hemolysis. After streaking the agar plate, the simplest technique to produce an anaerobic “pocket” is to “stab” the inoculating loop vertically into the agar. (As depicted in Figure 3, most strains of Streptococcus pyogenes also produce the oxygen-stable hemolysin “Streptolysin S,” which causes lysis in ambient air.)
Beta hemolysis on Blood Agar
Beta hemolysis on Blood Agar – (A) and (B) Normal Upper respiratory flora mixed with Streptococcus pyogenes demonstrating production of Streptolysin O. Beta hemolysis is only evident where the agar was “stabbed”.
  • Streptococcus agalactiae (Lancefield group B) and Listeria monocytogenes are more examples. For some species, the hemolysin may be generated very slowly or have a poor reactivity. The visible hemolysis may be so mild that it is only discernible immediately beneath the colony (rather than broadly diffused as in S. pyogenes, above). To visualise this extremely mild reaction, the colony can be removed via an inoculating loop, allowing one to observe the lysed cells just beneath where the colony had been developing. (See CAMP technique for additional information on Streptococcus agalactiae and numerous hemolysins.)
Beta hemolysis on Blood Agar
Beta hemolysis on Blood Agar – (A) Streptococcus agalactiae (Lancefield group B) viewed with incident light: No obvious hemolysis. (B) Streptococcus agalactiae (Lancefield group B) viewed with transmitted light: Subtle hemolysis. (C) Listeria monocytogenes, removing colonies to see the subtle pink hemolysis directly beneath the colonies

c. Gamma hemolysis on Blood Agar

  • Gamma hemolysis is also known as non-hemolysis because there is no hemolysis of red blood cells is observed.
  • In the end, there is no color change or a hemolysis-like zone is seen under or around the colonies.
  • The species like Neisseria meningiditis are not hemolytic or Gamma-hemolytic.
  • Gamma hemolysis (γ) is inconsistent with itself. Gamma represents the absence of hemolysis. No reaction should occur in the surrounding media.
Gamma hemolysis on Blood Agar
Gamma hemolysis on Blood Agar – (A) “Gamma Streptococcus” or Enterococcus faecalis (24 hours, non-hemolytic). “Gammastreptococcus” are usually non-hemolytic after 24 hours of incubation, but many eventually display weak alpha hemolysis. (The genus Enterococcus was once a part of the Streptococcus genus, and was considered a “gamma Streptococcus species”. Enterococci usually reacts as Lancefield group D.) (B) The same Enterococcus strain as Figure (A), shown with transmitted light at 48 hours incubation demonstrates the alpha

d. Alpha prime or wide zone alpha hemolysis

  • Alpha prime hemolysis can be defined as small areas of erythrocytes in good condition close to the colony of bacterial cells and includes the complete lysis of RBCs that surrounds the area of intact erythrocytes.
  • It could be confused with B-hemolysis due the presence of a clear area within the colonies.

e. Target Hemolysis

  • Clostridium perfringens is easily detected in the laboratory by its distinctive “double zone” hemolysis, sometimes referred to as target hemolysis.

Image of Blood agar plates – Hemolysis on blood agar

Blood Agar and Hemolysis
Blood agar plates – hemolysis on blood agar | Image Source: https://paramedicsworld.com

Preparation of Blood Agar

  1. Approximately 40 grammes of the prepared medium are added to 1000 millilitres of deionized or distilled water.
  2. The suspension is brought to a boil to completely dissolve the medium.
  3. It is then autoclaved at 15 pounds of pressure and 121 degrees Fahrenheit for approximately 15 minutes.
  4. The medium is then removed from the autoclave and chilled to 40 to 45 degrees Celsius.
  5. Aseptically, 5% v/v sterile defibrinated blood is added and thoroughly mixed.
  6. The media is subsequently poured under sterile conditions into sterile Petri dishes.
  7. Once the media has solidified, the plates can be placed in a hot air oven at a lower temperature setting for a few minutes to remove any moisture before use.

Storage and self-life of Blood Agar

  • The powdered media should be stored between 10 and 30°C in an airtight container, while the produced media should be stored between 20 and 30°C.
  • As the medium is hygroscopic in nature and therefore absorbs moisture rather fast, it should be stored appropriately after opening when dry and tightly sealed to prevent lump formation.
  • The container with the medium should be stored in a dry ventilated place shielded from extremes of temperature and sources of ignite.
  • The product should be utilised prior to the expiration date listed on the label.

Result Interpretation on Blood Agar

The basal medium appears light amber in hue and may appear transparent to somewhat opalescent. The addition of 5% v/v sterile defibrinated blood results in the formation of a cherry-red, opaque gel on the Petri plates. The following table illustrates the growth and colony morphologies of significant medicinal microorganisms on Blood Agar Medium:

OrganismGrowthColony MorphologyHemolysis
Neisseria meningiditis on Blood AgarGood-luxuriantGrey and unpigmented colonies that appear round, smooth, moist, glistening, and convex, with a clearly defined edge.Non-hemolytic or γ-hemolytic.
Salmonella Typhi on Blood AgarGood-luxuriantSmooth colorless colonies that are smooth, moist, and flat with a diameter range of 2-4 mm.Non-hemolytic or γ-hemolytic.
Staphylococcus aureus on Blood AgarLuxuriantGolden yellow colored circular, convex and smooth colonies of the diameter range of 2-4 mm; opaque colonies with a zone of hemolysis.β-hemolytic.
Staphylococcus epidermidis on Blood AgarLuxuriantCircular, colonies of the size 1-4 mm in diameter; grey to white-colored with low convex elevation; moist, glistening colonies.Non-hemolytic or γ-hemolytic.
Streptococcus pyogenes on Blood AgarLuxuriantWhite-greyish-colored colonies with a diameter of > 0.5 mm; the colonies are surrounded by a zone of β-hemolysis that is often two to four times as large as the colony diameter.β-hemolytic.
Streptococcus pneumonia on Blood AgarLuxuriantsmall, grey, moist (sometimes mucoidal in encapsulated virulent strains), colonies with the characteristic zone of alpha-hemolysis (green); due to autolysis, often produces a dimple-like zone of hemolysis than the typical crater-like appearance.α-hemolytic.
Pseudomonas aeruginosa on Blood AgarGood-luxuriantLarge colonies of the size 2-5mm in diameter; flat colonies that are grey to white-colored with an undulate margin with a zone of β-hemolysis.β-hemolytic.

Pseudomonas aeruginosa on blood agar

Pseudomonas aeruginosa is a gram-negative bacterium that can be found in a variety of environments, including soil, water, and plants. It is also a common pathogen that can cause infections in humans, particularly in people with compromised immune systems.

On blood agar, Pseudomonas aeruginosa typically appears as small, shiny, and round colonies that are surrounded by a halo of beta-hemolysis. Beta-hemolysis is the partial or complete lysis of red blood cells, and it can be seen as a clear zone around the colonies on blood agar. Pseudomonas aeruginosa is also a facultative anaerobe, which means that it can grow in the presence or absence of oxygen.

In addition to its characteristic appearance on blood agar, Pseudomonas aeruginosa can also be identified based on its biochemical properties. For example, it is a positive test for the production of the enzymes catalase and oxidase, and it is also a positive test for the production of fluorescein, which is a pigment that can be seen under UV light.

Overall, Pseudomonas aeruginosa is a common cause of infections in humans, and it is important to accurately identify this bacterium in order to initiate appropriate treatment.

Streptococcus pneumoniae blood agar

Streptococcus pneumoniae is a bacterium that can cause a variety of infections, including pneumonia, sinus infections, and ear infections. It is often cultured on blood agar plates in order to identify and study the bacterium. Blood agar is a type of agar plate that contains sheep blood, which allows for the growth of fastidious organisms like S. pneumoniae. The bacteria can be identified on the plate by its characteristic appearance and by the presence of certain enzymes and other metabolic products. Additionally, various biochemical tests can be performed on the culture to confirm the identity of the bacterium.

On a blood agar plate, Streptococcus pneumoniae typically appears as small, round, and white or cream-colored colonies. The colonies may appear translucent or slightly opaque, and they are usually surrounded by a clear zone. This clear zone is caused by the bacterium’s production of a substance called alpha-hemolysin, which breaks down the red blood cells in the agar and causes the blood to appear clear or greenish around the colony. In some cases, the colonies of S. pneumoniae may appear gray or green due to the production of a pigment called pneumolysin. The appearance of the colonies can vary depending on the specific strain of S. pneumoniae and the conditions of the culture.

Streptococcus pyogenes on blood agar

Streptococcus pyogenes is a bacterium that can cause a variety of infections, including strep throat, impetigo, and cellulitis. It is often cultured on blood agar plates in order to identify and study the bacterium. On a blood agar plate, S. pyogenes typically appears as small, round, and white or cream-colored colonies. The colonies may appear translucent or slightly opaque, and they are usually surrounded by a clear zone. This clear zone is caused by the bacterium’s production of a substance called alpha-hemolysin, which breaks down the red blood cells in the agar and causes the blood to appear clear or greenish around the colony. In some cases, the colonies of S. pyogenes may appear gray or green due to the production of a pigment called pyocyanin. The appearance of the colonies can vary depending on the specific strain of S. pyogenes and the conditions of the culture.

Staphylococcus aureus on blood agar

Staphylococcus aureus is a bacterium that can cause a variety of infections, including skin infections, respiratory infections, and food poisoning. It is often cultured on blood agar plates in order to identify and study the bacterium. On a blood agar plate, S. aureus typically appears as small, round, and yellow or golden-colored colonies. The colonies may appear shiny or glossy and are usually surrounded by a zone of clear or greenish-colored fluid. This fluid is caused by the bacterium’s production of a substance called alpha-hemolysin, which breaks down the red blood cells in the agar and causes the blood to appear clear or greenish around the colony. The appearance of the colonies can vary depending on the specific strain of S. aureus and the conditions of the culture.

Enterococcus faecalis blood agar

Enterococcus faecalis is a gram-positive bacterium that is commonly found in the human intestinal tract and is also present in the environment. It is often used as a model organism in laboratory research. When grown on blood agar, E. faecalis can produce small, circular, green-black colonies with a metallic sheen. It is a facultative anaerobe, meaning that it can grow in the presence or absence of oxygen, but it grows better in the presence of oxygen. E. faecalis is resistant to many antibiotics, including penicillins, making it an important pathogen in the hospital setting. It can cause a range of infections, including urinary tract infections, endocarditis, and wound infections.

Quality control

  • Inoculate a prepared blood agar with Streptococcus pneumoniae and Streptococcus pyogenes.
  • Observation of Cultural properties after 18-24 hours of incubation at 35-37°C with 7% v/v sterile sheep blood.
OrganismGrowthHaemolysis
Streptococcus pneumoniaeluxuriantalpha
Streptococcus pyogenesluxuriantBeta

Streak-stab technique

In order to better see hemoloysis patterns, researchers will often puncture multiple holes in the agar with an inoculating loop when inoculating a BAP. A specific hemolysin produced by Streptococcus pyogenes, streptolysin O, can be detected with this jab. Hemolysin is deactivated by oxygen and is only detected below the surface (in anaerobic conditions) around the site of the wound. Streptolysin O is responsible for the oval-shaped clearance around the puncture wounds in the image below.

Streak-stab technique
Streak-stab technique

Types of Blood Agar

1. Blood agar plate

  • Blood agar plates (BAPs) typically contain 5–10% concentrations of mammalian blood (often that of sheep or horse). BAPs are enriched, differentiated media used to separate difficult-to-cultivate organisms and detect their hemolytic activity.
  • The lysis and complete digesting of red blood cell contents surrounding a colony will be evidenced by β-hemolytic activity. Among the examples is Streptococcus haemolyticus. Due to the conversion of haemoglobin to methemoglobin, α-hemolysis will only induce a partial lysis of the red blood cells (the cell membrane will remain intact) and will result in a green or brown hue. This is illustrated by Streptococcus viridans.
  • γ-Hemolysis (or nonhemolytic) describes the absence of hemolytic activity. Additionally, BAPs include beef extract, tryptone, sodium chloride, and agar.

2. Chocolate agar

  • Blood cells are lysed on chocolate agar by heating the plate to 80 degrees Celsius. It is used to cultivate finicky respiratory bacteria like Haemophilus influenzae. Chocolate agar gets its name from its colour, however it contains no genuine chocolate.

3. Horse blood agar

  • Horse blood agar is a form of microbiological culture medium containing blood. As it is enriched, it permits the growth of specific finicky bacteria and enables the detection of hemolytic activity in these cultures.

4. Thayer–Martin agar

  • Thayer–Martin agar is developed to isolate Neisseria gonorrhoeae.

5. Thiosulfate–citrate–bile salts–sucrose agar

  • Thiosulfate–citrate–bile salts–sucrose agar promotes growth of Vibrio spp., including Vibrio cholerae.

Application of Blood Agar

  • Blood agar is used primarily for the culture and isolation of finicky organisms such as Neisseria and Streptococcus.
  • They can also be used to distinguish between α-, β- or γ-hemolytic bacterial hemolysis on agar plates.
  • If phenolphthalein phosphate is added to blood agar, then phosphate-producing Staphylococci can be identified.
  • Antigens from Salmonella Typhi are commonly prepared on blood agar.
  • The blood agar base technique is widely used in the food science industry.

Limitations of Blood Agar

  • Blood agar inhibits the development of Haemophilus hemolyticus due to the presence of several inhibitors that can only be deactivated by heating the medium after the addition of blood.
  • The pattern of hemolysis may vary depending on the blood utilised.
  • The addition of rabbit or horse blood to the culture media promotes the growth of H. hemolyticus; nonetheless, the growth resembles that of Streptococcus species and must be validated.

Greening reaction on blood agar??

It is possible for bacteria growing on blood agar to produce a greening reaction, which is the production of a green pigment called pyocyanin. This pigment is produced by certain species of bacteria, including Pseudomonas aeruginosa and some strains of Staphylococcus aureus. The greening reaction is often seen when these bacteria are grown on blood agar that has been incubated at room temperature for an extended period of time. The green color is produced by the oxidation of the pyocyanin pigment, which can occur when the bacteria are exposed to oxygen.

In some cases, the greening reaction may be accompanied by the production of other pigments, such as pyoverdin, which is a fluorescent green pigment produced by Pseudomonas aeruginosa. The presence of these pigments can be used to help identify specific bacterial species and can also provide information about the metabolic activity of the bacteria.

Important Notes

  • Blood is an excellent component of enriched medium for fussy organisms, despite the fact that it includes inhibitors for specific bacterial genera, such as Neisseria and Haemophilus, and that blood agar must be heated to deactivate these inhibitors and release critical growth factors (e.g., V factor). Heating blood agar transforms it into chocolate agar (at 75°C for 15 minutes, it turns chocolate-colored) and promotes the development of these bacteria.
  • Hemolysis on blood agar: Streptococci cause primarily three types of hemolysis in Sheep blood agar: Alpha hemolysis, Beta hemolysis, and gamma hemolysis; however, alpha prime or wide zone alpha hemolysis may also occur. Hemolysis is most easily noticed by studying colonies formed in anaerobic circumstances or by viewing colonies beneath the surface. How can one determine whether the colonies observed on a plate caused alpha, beta, or gamma hemolysis? – Principle and interpretation should be followed.
  • To determine the type of blood agar hemolysis, the plate must be put up to a source of bright transmitted light and inspected with the light coming from behind.
  • Streptococcus pneumoniae has an alpha hemolysis.
  • Group A beta-hemolytic streptococci—Streptococcus pyogenes and Group B beta-hemolytic streptococci—Streptococcus agalactiace are beta-hemolytic streptococci. For group A streptococci, the maximum activity of both oxygen-labile SLO and oxygen-stable SLS hemolysins is exclusively detected under anaerobic circumstances.
  • Gamma or lack of hemolysis: Enterococcus species
  • Alpha prime or wide zone alpha hemolysis: A tiny zone of undamaged erythrocytes immediately close to the bacterial colony, surrounded by a zone of complete red-cell hemolysis. This form of hemolysis can be mistaken for beta hemolysis.
  • Some organisms, such as Clostridium perfringens and Aeromonas hydrophilia, exhibit what is known as target hemolysis, which is characterised by a twofold zone of hemolysis on its surface.
  • Prepare a small number of blood agar plates before preparing a large quantity to check that the blood is sterile.
  • Streptococcus agalactiae CAMP test. -positive blood agar as demonstrated below-

Blood Agar Plate Results – Video

Staphylococcus aureus on blood agar

Streptococcus pyogenes on blood agar

Streptococcus pneumoniae blood agar

FAQ

Is blood agar selective or differential?

Blood agar is a type of culture medium that is used to grow and isolate microorganisms, particularly bacteria. It is made by adding blood to a base agar medium, which is a solidifying agent that is commonly used in microbiology. Blood agar can be either selective or differential, depending on the specific formulation of the medium.
Selective media are designed to favor the growth of certain types of microorganisms while inhibiting the growth of others. For example, blood agar that contains antibiotics such as penicillin can be selective for bacteria that are resistant to the antibiotic.
Differential media are designed to differentiate between different types of microorganisms based on their biochemical characteristics. For example, blood agar that contains an indicator such as phenol red can be differential for bacteria that produce hydrogen sulfide, because these bacteria will cause the indicator to change color.
In general, blood agar is considered to be both selective and differential because it contains nutrients that can support the growth of many types of bacteria, as well as indicators that can be used to differentiate between different types of bacteria based on their biochemical properties.

Why is blood agar useful as a primary isolation medium?

Blood agar is a type of agar media that is used for the primary isolation of bacteria. It is a general-purpose medium that can be used to grow a wide variety of bacteria, including both aerobes and anaerobes. One of the main advantages of blood agar is that it contains red blood cells, which can be used as a source of nutrients for the bacteria. This makes it an excellent medium for growing fastidious bacteria, which are bacteria that have high nutritional requirements and are difficult to grow in other media.
In addition, blood agar can be used to differentiate between different types of bacteria based on their ability to hemolyze (break down) the red blood cells. Some bacteria produce enzymes that can break down the red blood cells, resulting in a zone of clearing around the bacterial colony on the agar. This is known as hemolysis. Different types of hemolysis, such as alpha hemolysis and beta hemolysis, can be used to identify specific bacterial species.
Overall, blood agar is an important tool in microbiology laboratories because it allows for the cultivation and identification of a wide range of bacteria.

What does blood agar test for?

Blood agar is a type of agar media that is used to isolate and identify bacterial species. It can be used to test for the presence of various types of bacteria, including both aerobes and anaerobes.
One of the main uses of blood agar is to determine the ability of bacteria to grow in the presence of red blood cells. The red blood cells in the agar provide a source of nutrients for the bacteria, and the presence of bacterial growth on the agar indicates that the bacteria are able to utilize these nutrients.
Blood agar can also be used to differentiate between different types of bacteria based on their ability to hemolyze (break down) the red blood cells. Some bacteria produce enzymes that can break down the red blood cells, resulting in a zone of clearing around the bacterial colony on the agar. This is known as hemolysis. Different types of hemolysis, such as alpha hemolysis and beta hemolysis, can be used to identify specific bacterial species.
Overall, blood agar is an important tool in microbiology laboratories because it allows for the cultivation and identification of a wide range of bacteria.

what bacteria grows on blood agar?

Blood agar is a general-purpose medium that can be used to grow a wide variety of bacteria. Many different types of bacteria are able to grow on blood agar, including both aerobes and anaerobes. Some examples of bacteria that can grow on blood agar include:
Streptococcus species
Staphylococcus species
Enterococcus species
Haemophilus species
Neisseria species
Bordetella species
Escherichia coli
Salmonella species
Shigella species
Pseudomonas aeruginosa

This is by no means an exhaustive list, as many other types of bacteria can also grow on blood agar. The specific types of bacteria that grow on the medium will depend on the conditions in which it is incubated, as well as the bacterial species present in the sample being tested.

Do all bacteria grow on blood agar?

Not all bacteria are able to grow on blood agar. Blood agar is a general-purpose medium that can support the growth of many different types of bacteria, including both aerobes and anaerobes. However, some bacteria may not be able to utilize the nutrients provided by the red blood cells in the agar, and as a result, may not grow well or may not grow at all on this medium.
Additionally, some bacteria may produce toxins or enzymes that can break down the red blood cells in the agar, resulting in reduced growth or no growth on the medium.
It is important to note that the specific types of bacteria that are able to grow on blood agar will depend on the conditions in which it is incubated, as well as the bacterial species present in the sample being tested.

Why is it called blood agar?

Blood agar is called blood agar because it contains red blood cells. Agar is a type of polysaccharide that is derived from algae and is used as a solidifying agent in the preparation of media for cultivating microorganisms. When agar is combined with blood, it forms a solid gel that is known as blood agar.
The red blood cells in the blood agar serve as a source of nutrients for the bacteria being cultured. They provide a rich source of proteins, lipids, and other nutrients that are essential for the growth and metabolism of many bacterial species. The blood also serves as a good indicator of the ability of the bacteria to utilize the nutrients in the media, as the presence of bacterial growth on the agar indicates that the bacteria are able to utilize the nutrients provided.
In addition to its use as a nutrient source, the blood in blood agar can also be used to differentiate between different types of bacteria based on their ability to hemolyze (break down) the red blood cells. This is why blood agar is an important tool in microbiology laboratories for the cultivation and identification of a wide range of bacteria.

What are the 3 types of hemolysis?

There are three types of hemolysis that can be observed when growing bacteria on blood agar: alpha hemolysis, beta hemolysis, and gamma hemolysis. These terms refer to the ability of the bacteria to break down the red blood cells in the agar, resulting in a zone of clearing around the bacterial colony.
1. Alpha hemolysis: Alpha hemolysis is a type of partial hemolysis that results in the production of a greenish discoloration of the agar surrounding the bacterial colony. This is caused by the production of enzymes that partially break down the red blood cells, releasing the hemoglobin into the agar.

2. Beta hemolysis: Beta hemolysis is a type of complete hemolysis that results in the complete breakdown of the red blood cells, producing a clear zone around the bacterial colony. This is caused by the production of enzymes that completely lyse (break down) the red blood cells.

3. Gamma hemolysis: Gamma hemolysis, also known as nonhemolytic, refers to the lack of hemolysis. In this case, the red blood cells remain intact, and no zone of clearing is observed around the bacterial colony.
The type of hemolysis observed can be used to differentiate between different bacterial species and can also provide information about the metabolic activity of the bacteria.

Is blood agar used for E coli?

Yes, blood agar can be used to isolate and identify Escherichia coli (E. coli). E. coli is a gram-negative bacterium that is commonly found in the human intestinal tract and is also present in the environment. It is an important indicator of fecal contamination and is often used as a marker for the presence of other pathogenic bacteria.
When grown on blood agar, E. coli typically produces small, circular, pink-red colonies with a moist, shiny appearance. It is a facultative anaerobe, meaning that it can grow in the presence or absence of oxygen, but it grows better in the presence of oxygen. E. coli is typically sensitive to a wide range of antibiotics, making it easier to treat than some other bacterial infections. However, some strains of E. coli are resistant to certain antibiotics and can cause serious infections, particularly in people with compromised immune systems.
Overall, blood agar is a useful medium for the cultivation and identification of E. coli and many other types of bacteria.

Why is blood agar used for Staphylococcus aureus?

Staphylococcus aureus is a gram-positive bacterium that is commonly found on the skin and in the nasal passages of humans and animals. It is an important pathogen that can cause a range of infections, including skin infections, wound infections, respiratory tract infections, and food poisoning.

Blood agar is often used to isolate and identify S. aureus because it is a general-purpose medium that can support the growth of a wide range of bacteria, including both aerobes and anaerobes. When grown on blood agar, S. aureus typically produces small, circular, yellow-gold colonies with a smooth, shiny appearance. It is a facultative anaerobe, meaning that it can grow in the presence or absence of oxygen, but it grows better in the presence of oxygen.

In addition to its use for the cultivation of S. aureus, blood agar can also be used to differentiate between different types of S. aureus based on their ability to produce certain enzymes or pigments. For example, some strains of S. aureus are able to produce the enzyme coagulase, which causes the blood in the agar to coagulate (clot). The presence of this enzyme can be used to distinguish S. aureus from other types of staphylococci.

Overall, blood agar is an important tool in microbiology laboratories for the cultivation and identification of S. aureus and many other types of bacteria.

Which factor does blood agar have?

Blood agar is a type of agar media that contains red blood cells suspended in a nutrient-rich medium. The red blood cells provide a source of nutrients for the bacteria being cultured, including proteins, lipids, and other essential nutrients. The blood also contains growth factors, such as vitamins and minerals, which are necessary for the growth and metabolism of many bacterial species.

In addition to the nutrients provided by the blood, blood agar also contains other factors that are important for the growth of bacteria. These include:

Agar: Agar is a type of polysaccharide that is derived from algae and is used as a solidifying agent in the preparation of media for cultivating microorganisms. It provides a solid matrix for the bacteria to grow on and also serves as a source of nutrients.
pH: Blood agar is usually prepared with a pH of around 7.2-7.4, which is neutral to slightly alkaline. This pH range is suitable for the growth of many bacterial species.
Oxygen: Blood agar is typically incubated in the presence of oxygen, which allows for the growth of aerobic bacteria. Some bacteria, such as obligate aerobes, require the presence of oxygen to grow.
Temperature: Blood agar is typically incubated at a temperature of 35-37°C, which is optimal for the growth of many bacterial species.

Overall, the combination of these factors in blood agar makes it a suitable medium for the cultivation and identification of a wide range of bacteria.

Does E coli grow on blood agar?

Yes, Escherichia coli (E. coli) can grow on blood agar. E. coli is a gram-negative bacterium that is commonly found in the human intestinal tract and is also present in the environment. It is an important indicator of fecal contamination and is often used as a marker for the presence of other pathogenic bacteria.
When grown on blood agar, E. coli typically produces small, circular, pink-red colonies with a moist, shiny appearance. It is a facultative anaerobe, meaning that it can grow in the presence or absence of oxygen, but it grows better in the presence of oxygen. E. coli is typically sensitive to a wide range of antibiotics, making it easier to treat than some other bacterial infections. However, some strains of E. coli are resistant to certain antibiotics and can cause serious infections, particularly in people with compromised immune systems.
Overall, blood agar is a useful medium for the cultivation and identification of E. coli and many other types of bacteria.

What is another name for hemolysis?

Hemolysis, also spelled haemolysis and referred to as hematolysis, is the disintegration or death of red blood cells, releasing the oxygen-carrying pigment haemoglobin into the surrounding media.

Is blood agar selective for Gram positive?

Blood agar is not inherently selective for Gram-positive bacteria. In other words, it does not have any properties that inhibit the growth of Gram-negative bacteria or promote the growth of Gram-positive bacteria.
However, blood agar can be made selective for Gram-positive bacteria by the addition of certain agents, such as bacitracin or vancomycin. These agents inhibit the growth of Gram-negative bacteria, while allowing Gram-positive bacteria to grow.
Alternatively, blood agar can be made selective for Gram-negative bacteria by the addition of certain agents, such as polymyxin B or anionic detergents (e.g., sodium lauryl sulfate). These agents inhibit the growth of Gram-positive bacteria, while allowing Gram-negative bacteria to grow.
Overall, the selectivity of blood agar depends on the specific additives that are used in its preparation.

Can Gram positive bacteria grow on blood agar?

Yes, Gram-positive bacteria can grow on blood agar. In fact, many Gram-positive bacteria are able to utilize the nutrients in blood agar for growth, and they may also produce enzymes that can break down red blood cells (hemolysins), leading to the production of visible halos on the agar surface.
However, it is important to note that the growth of Gram-positive bacteria on blood agar may be affected by the presence of certain agents that are added to the agar during its preparation. For example, the addition of bacitracin or vancomycin can make blood agar selective for Gram-positive bacteria, while the addition of polymyxin B or anionic detergents (e.g., sodium lauryl sulfate) can make blood agar selective for Gram-negative bacteria.
Overall, the ability of Gram-positive bacteria to grow on blood agar depends on the specific additives that are used in its preparation, as well as the growth requirements of the individual bacterial species.

Does blood agar show Gram positive or negative?

Blood agar does not inherently show whether a bacterium is Gram-positive or Gram-negative. In other words, it does not have any properties that specifically stain or differentiate between these two types of bacteria.
Gram staining is a laboratory technique that is used to differentiate between Gram-positive and Gram-negative bacteria based on the chemical and physical properties of their cell walls. In order to perform Gram staining, bacteria are first grown on a solid media such as blood agar, and then a series of steps are followed to apply crystal violet and iodine to the bacterial cells, decolorize them with alcohol or acetone, and counterstain them with safranin.
Based on the results of Gram staining, bacteria are classified as either Gram-positive (if they retain the crystal violet-iodine complex and appear purple or blue under the microscope) or Gram-negative (if they lose the crystal violet-iodine complex and appear pink or red under the microscope).
Overall, blood agar is a useful media for growing bacteria, but it is not used for the purpose of Gram staining or differentiation between Gram-positive and Gram-negative bacteria.

Does salmonella grow on blood agar?

Yes, Salmonella can grow on blood agar. Blood agar is a type of nutrient agar that is made by adding heat-inactivated blood (usually from a sheep or horse) to a base of nutrient agar. The blood serves as a source of nutrients for the bacteria, and it also helps to stimulate bacterial growth.
Salmonella is a type of Gram-negative bacteria that can cause a wide range of illnesses, including food poisoning, typhoid fever, and paratyphoid fever. It is an aerobic bacterium, meaning that it requires oxygen to grow and survive.
When grown on blood agar, Salmonella typically appears as small, pink-white or gray-white, circular colonies with a smooth, glossy surface. It may also produce a pitting or dimpling effect on the agar surface, known as “satellite colonies.”
Overall, blood agar is a useful media for the cultivation and identification of Salmonella in the laboratory.

Why is blood agar used when culturing bacteria?

Blood agar is a type of nutrient agar that is used to culture bacteria. It is made by adding heat-inactivated blood (usually from a sheep or horse) to a base of nutrient agar. The blood serves as a source of nutrients for the bacteria, and it also helps to stimulate bacterial growth.

There are several reasons why blood agar is used when culturing bacteria:

  1. It allows for the identification of bacteria that produce enzymes that can break down red blood cells (hemolysins), leading to the production of visible halos on the agar surface.
  2. It allows for the identification of bacteria that can utilize certain sugars, leading to the production of acid or gas.
  3. It allows for the differentiation of bacteria based on their ability to grow at different temperatures.
  4. It allows for the differentiation of bacteria based on their ability to grow in the presence or absence of oxygen.

Overall, blood agar is a useful tool for the identification and characterization of bacteria in the laboratory.

References

  • https://www.wikilectures.eu/w/Blood_agar
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Citation

APA

MN Editors. (November 2, 2021).Blood Agar Definition, Preparation, Composition, Application, and limitation.. Retrieved from https://microbiologynote.com/blood-agar/

MLA

MN Editors. "Blood Agar Definition, Preparation, Composition, Application, and limitation.." Microbiology Note, Microbiologynote.com, November 2, 2021.

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