Colonial morphology

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Colonial morphology of various specimens of Pseudomonas aeruginosa, including mucoid types Pseudomonas aeruginosa on blood agar.jpg
Colonial morphology of various specimens of Pseudomonas aeruginosa , including mucoid types

In microbiology, colonial morphology refers to the visual appearance of bacterial or fungal colonies on an agar plate. Examining colonial morphology is the first step in the identification of an unknown microbe. The systematic assessment of the colonies' appearance, focusing on aspects like size, shape, colour, opacity, and consistency, provides clues to the identity of the organism, allowing microbiologists to select appropriate tests to provide a definitive identification.

Contents

Procedure

Some terms used to describe colonial morphology Bacterial colony morphology.png
Some terms used to describe colonial morphology

When a specimen arrives in the microbiology laboratory, it is inoculated into an agar plate and placed in an incubator to encourage microbial growth. Because the appearance of microbial colonies changes as they grow, colonial morphology is examined at a specific time after the plate is inoculated. Usually, the plate is read at 18–24 hours post-inoculation, [1] : 163  but times may differ for slower-growing organisms like fungi. [2] : 518  The microbiologist examines the appearance of the colony, noting specific features such as size, colour, shape, consistency, and opacity. [1] : 165–8  A hand lens or magnifying glass may be used to view colonies in greater detail. [3] : 96 

The opacity of a microbial colony can be described as transparent, translucent, or opaque. Staphylococci are usually opaque, [1] : 167–8  while many Streptococcus species are translucent. [4] : 188  The overall shape of the colony may be characterized as circular, irregular, or punctiform (like pinpoints). The vertical growth or elevation of the colony, another identifying characteristic, is assessed by tilting the agar plate to the side and is denoted as flat, raised, convex, pulvinate (very convex), umbilicate (having a depression in the centre) or umbonate (having a bump in the centre). [1] : 167  [5] : 19  The edge of the colony may be separately described using terms like smooth, rough, irregular and filamentous. Bacillus anthracis is notable for its filamentous appearance, which is sometimes described as resembling Medusa's head. [1] : 167 

Consistency is examined by physically manipulating the colony with a sterile instrument. It is described using terms like brittle, creamy, sticky and dry. Staphylococci are considered to have a creamy consistency, [1] : 173  while some Neisseria species are sticky, and colonies of diphtheroid bacteria and beta-hemolytic streptococci are typically dry. [1] : 167–8  Bacteria that produce capsules often have a slimy (mucoid) consistency. [2] : 495 

When certain microorganisms are grown on blood agar, they may digest the blood in the medium, causing visible hemolysis (destruction of red blood cells) on the agar plate. In colonial morphology, hemolysis is classified into three types: alpha-, beta-, and gamma-hemolysis. In alpha-hemolysis, the blood is partially digested, causing the area around the colony to turn green. In beta-hemolysis, the organism digests the blood completely, leaving a clear area around each colony. [1] : 165–6  Organisms that do not produce hemolysis are referred to as gamma-hemolytic. [2] : 500  Clostridium perfringens , which causes gas gangrene, is noteworthy for producing a "double zone" of both complete and incomplete hemolysis. [6] : 94 

The odour of a culture is sometimes considered part of colonial morphology. While intentionally smelling microbial cultures is not advised, some organisms produce distinctive odours that can be detected during routine examination of the culture. Among these are Pseudomonas aeruginosa , which has a grape-like scent; Staphylococcus aureus , which is said to smell like old socks; and Proteus mirabilis , whose scent is alternately described as putrid [1] : 168  or like chocolate cake. [7] : 124 

Other distinctive features of colonial morphology include motility and the production of pigments. Pseudomonas aeruginosa produces the pigments pyocyanin and pyoverdin, which give the colonies a greenish sheen. [1] : 473  [5] : 154  Some specimens of Serratia marcescens produce an orange-red pigment called prodigiosin. [5] : 26  [8] : 236  Organisms with swarming motility, like Proteus species, exhibit concentric waves of growth extending from the inoculation point. [1] : 423  [5] : 153 

Interpretation

Colonial morphology serves as the first step in the identification of microbial species from clinical samples. [10] Based on the visual appearance of the colonies, microbiologists can narrow down the list of possible organisms, allowing them to select appropriate tests to provide a definitive diagnosis. For example, if a microbiologist observes colonies that resemble a Staphylococcus species, they may perform a catalase test to confirm that it belongs to the genus Staphylococcus, and a coagulase test to determine whether it is a coagulase-negative staphylococcus or a more pathogenic species, such as S. aureus. [3] : 101  [8] : 203 

Observation of hemolysis is useful in the presumptive identification of bacteria, [1] : 165–6  especially streptococci, which are classified on the basis of their hemolytic reactions. [11] : 92  For example, Streptococcus pyogenes , which causes strep throat and scarlet fever, [12] displays beta-hemolysis, while Streptococcus pneumoniae , which can cause pneumonia and meningitis, displays alpha-hemolysis. [2] : 507  The highly pathogenic S. aureus classically displays beta-hemolysis, [5] : 26  while Staphylococcus epidermidis , part of the normal skin flora and an occasional opportunistic pathogen, does so weakly or not at all. [1] : 309–14 

Although automated techniques like MALDI-TOF are increasingly used to identify microorganisms in clinical laboratories, colonial morphology remains useful to distinguish potential pathogens, which must be identified, from normal flora, for which definitive identification is unnecessary, [1] : 163  and to confirm identification when automated techniques give inconclusive results. [3] : 103–4 

Related Research Articles

<i>Streptococcus</i> Genus of bacteria

Streptococcus is a genus of gram-positive coccus or spherical bacteria that belongs to the family Streptococcaceae, within the order Lactobacillales, in the phylum Bacillota. Cell division in streptococci occurs along a single axis, so as they grow, they tend to form pairs or chains that may appear bent or twisted. This differs from staphylococci, which divide along multiple axes, thereby generating irregular, grape-like clusters of cells. Most streptococci are oxidase-negative and catalase-negative, and many are facultative anaerobes.

Agar plate Petri dish with agar used to culture microbes

An agar plate is a Petri dish that contains a growth medium solidified with agar, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics.

Methicillin Antibiotic medication

Methicillin, also known as meticillin, is a narrow-spectrum β-lactam antibiotic of the penicillin class.

Microbiological culture Method of allowing microorganisms to multiply in a controlled medium

A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are foundational and basic diagnostic methods used as a research tool in molecular biology.

Coccus Round shape of certain bacteria or archaea

A coccus is any bacterium or archaeon that has a spherical, ovoid, or generally round shape. Bacteria are categorized based on their shapes into three classes: cocci (spherical-shaped), bacillus (rod-shaped) and spirochetes (spiral-shaped) cells. Coccus refers to the shape of the bacteria, and can contain multiple genera, such as staphylococci or streptococci. Cocci can grow in pairs, chains, or clusters, depending on their orientation and attachment during cell division. In contrast to many bacilli-shaped bacteria, most cocci bacteria do not have flagella and are non-motile.

Viridans streptococci

The viridans streptococci are a large group of commensal streptococcal Gram-positive bacteria species that are α-hemolytic, producing a green coloration on blood agar plates, although some species in this group are actually γ-hemolytic, meaning they produce no change on blood agar. The pseudo-taxonomic term "Streptococcus viridans" is often used to refer to this group of species, but writers who do not like to use the pseudotaxonomic term prefer the terms viridans streptococci, viridans group streptococci (VGS), or viridans streptococcal species.

Blood culture Test to detect bloodstream infections

A blood culture is a medical laboratory test used to detect bacteria or fungi in a person's blood. Under normal conditions, the blood does not contain microorganisms: their presence can indicate a bloodstream infection such as bacteremia or fungemia, which in severe cases may result in sepsis. By culturing the blood, microbes can be identified and tested for resistance to antimicrobial drugs, which allows clinicians to provide an effective treatment.

Growth medium Solid, liquid or gel used to grow microorganisms or cells

A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss Physcomitrella patens. Different types of media are used for growing different types of cells.

Hemolysis (microbiology)

Hemolysis is the breakdown of red blood cells. The ability of bacterial colonies to induce hemolysis when grown on blood agar is used to classify certain microorganisms. This is particularly useful in classifying streptococcal species. A substance that causes hemolysis is a hemolysin.

A colony-forming unit is a unit used in microbiology. It estimates the number of bacteria or fungal cells in a sample which are viable, able to multiply via binary fission under the controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead. The visual appearance of a colony in a cell culture requires significant growth, and when counting colonies it is uncertain if the colony arose from one cell or a group of cells. Expressing results as colony-forming units reflects this uncertainty.

Streaking (microbiology) Method for isolation of bacterial strains

In microbiology, streaking is a technique used to isolate a pure strain from a single species of microorganism, often bacteria. Samples can then be taken from the resulting colonies and a microbiological culture can be grown on a new plate so that the organism can be identified, studied, or tested.

Mannitol salt agar

Mannitol salt agar or MSA is a commonly used selective and differential growth medium in microbiology. It encourages the growth of a group of certain bacteria while inhibiting the growth of others. . It contains a high concentration of salt (NaCl) which is inhibitory to most bacteria - making MSA selective against most Gram-negative and selective for some Gram-positive bacteria that tolerate high salt concentrations. It is also a differential medium for mannitol-fermenting staphylococci, containing carbohydrate mannitol and the indicator phenol red, a pH indicator for detecting acid produced by mannitol-fermenting staphylococci. Staphylococcus aureus produces yellow colonies with yellow zones, whereas other coagulase-negative staphylococci produce small pink or red colonies with no colour change to the medium. If an organism can ferment mannitol, an acidic byproduct is formed that causes the phenol red in the agar to turn yellow. It is used for the selective isolation of presumptive pathogenic (pp) Staphylococcus species.

Petrifilm Plating system developed by 3M

The 3M Petrifilm plate is an all-in-one plating system made by the Food Safety Division of the 3M Company. They are heavily used in many microbiology-related industries and fields to culture various micro-organisms and are meant to be a more efficient method for detection and enumeration compared to conventional plating techniques. A majority of its use is for the testing of foodstuffs.

Etest

Etest is a way of determining antimicrobial sensitivity by placing a strip impregnated with antimicrobials onto an agar plate. A strain of bacterium or fungus will not grow near a concentration of antibiotic or antifungal if it is sensitive. For some microbial and antimicrobial combinations, the results can be used to determine a minimum inhibitory concentration (MIC). Etest is a proprietary system manufactured by bioMérieux. It is a laboratory test used in healthcare settings to help guide physicians by indicating what concentration of antimicrobial could successfully be used to treat patients' infections.

CAMP test

The CAMP test (Christie–Atkins–Munch-Peterson) is a test to identify group B β-hemolytic streptococci based on their formation of a substance that enlarges the area of hemolysis formed by the β-hemolysin elaborated from Staphylococcus aureus.

Staphylococcus schleiferi is a Gram-positive, cocci-shaped bacterium of the family Staphylococcaceae. It is facultatively anaerobic, coagulase-variable, and can be readily cultured on blood agar where the bacterium tends to form opaque, non-pigmented colonies and beta (β) hemolysis. There exists two subspecies under the species S. schleiferi: Staphylococcus schleiferi subsp. schleiferi and Staphylococcus schleiferi subsp. coagulans.

Staphylococcus pseudintermedius is a gram positive coccus bacteria of the genus Staphylococcus found worldwide. It is primarily a pathogen for domestic animals, but has been known to affect humans as well.S. pseudintermedius is an opportunistic pathogen that secretes immune modulating virulence factors, has many adhesion factors, and the potential to create biofilms, all of which help to determine the pathogenicity of the bacterium. Diagnoses of Staphylococcus pseudintermedius have traditionally been made using cytology, plating, and biochemical tests. More recently, molecular technologies like MALDI-TOF, DNA hybridization and PCR have become preferred over biochemical tests for their more rapid and accurate identifications. This includes the identification and diagnosis of antibiotic resistant strains.

Granada medium

Granada medium is a selective and differential culture medium designed to selectively isolate Streptococcus agalactiae and differentiate it from other microorganisms. Granada Medium was developed by Dr. Manuel Rosa-Fraile et al. at the Service of Microbiology in the Hospital Virgen de las Nieves in Granada (Spain).

In microbiology, the term isolation refers to the separation of a strain from a natural, mixed population of living microbes, as present in the environment, for example in water or soil flora, or from living beings with skin flora, oral flora or gut flora, in order to identify the microbe(s) of interest. Historically, the laboratory techniques of isolation first developed in the field of bacteriology and parasitology, before those in virology during the 20th century.

Diagnostic microbiology is the study of microbial identification. Since the discovery of the germ theory of disease, scientists have been finding ways to harvest specific organisms. Using methods such as differential media or genome sequencing, physicians and scientists can observe novel functions in organisms for more effective and accurate diagnosis of organisms. Methods used in diagnostic microbiology are often used to take advantage of a particular difference in organisms and attain information about what species it can be identified as, which is often through a reference of previous studies. New studies provide information that others can reference so that scientists can attain a basic understanding of the organism they are examining.

References

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  6. ↑ Apurba Sankar Sastry; Bhat Sandhya K (20 August 2017). Essentials of Practical Microbiology. JP Medical Ltd. ISBN   978-93-5270-185-8.
  7. ↑ Luis M. de la Maza; Marie T. Pezzlo; Cassiana E. Bittencourt; Ellena M. Peterson (16 June 2020). Color Atlas of Medical Bacteriology. John Wiley & Sons. ISBN   978-1-68367-035-3.
  8. 1 2 3 4 5 Karen C. Carroll; Janet S. Butel; Stephen A. Morse (12 August 2015). Jawetz Melnick & Adelbergs Medical Microbiology 27 E. McGraw-Hill Education. ISBN   978-0-07-182503-0.
  9. ↑ M. R. Adams; M. O. Moss (2008). Food Microbiology. Royal Society of Chemistry. ISBN   978-0-85404-284-5.
  10. ↑ Bae, Euiwon; Kim, Huisung; Rajwa, Bartek; Thomas, John G.; Robinson, J. Paul (2015). "Current status and future prospects of using advanced computer-based methods to study bacterial colonial morphology". Expert Review of Anti-infective Therapy. 14 (2): 207–218. doi:10.1586/14787210.2016.1122524. ISSN   1478-7210. PMID   26582139. S2CID   38205341.
  11. ↑ Karin C. VanMeter; Robert J. Hubert; William G. VanMeter (7 August 2013). Microbiology for the Healthcare Professional. Elsevier Health Sciences. ISBN   978-0-323-27702-0.
  12. ↑ Androulla, Efstratiou; Theresa, Lamagni (10 February 2016). "Epidemiology of Streptococcus pyogenes". Streptococcus pyogenes : Basic Biology to Clinical Manifestations. Oklahoma City, United States: University of Oklahoma Health Sciences Center. Retrieved 6 March 2020.

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