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A flowchart. A square plate with a grid of circles is labeled grow microorganisms on MALDI-plate. Then irradiate sample. Then use mass spectrometer to measure gaseous ions released. Then compare mass spectrum of sample to reference spectra; two small graphs are shown here. Then identify specied.
MALDI-TOF methods are now routinely used for diagnostic procedures in clinical microbiology laboratories. This technology is able to rapidly identify some microorganisms that cannot be readily identified by more traditional methods. (credit “MALDI plate photo”: modification of work by Chen Q, Liu T, Chen G; credit “graphs”: modification of work by Bailes J, Vidal L, Ivanov DA, Soloviev M)

Microbes can also be identified by measuring their unique lipid profiles. As we have learned, fatty acids of lipids can vary in chain length, presence or absence of double bonds, and number of double bonds, hydroxyl groups, branches, and rings. To identify a microbe by its lipid composition, the fatty acids present in their membranes are analyzed. A common biochemical analysis used for this purpose is a technique used in clinical, public health, and food laboratories. It relies on detecting unique differences in fatty acids and is called fatty acid methyl ester (FAME) analysis . In a FAME analysis , fatty acids are extracted from the membranes of microorganisms, chemically altered to form volatile methyl esters, and analyzed by gas chromatography (GC) . The resulting GC chromatogram is compared with reference chromatograms in a database containing data for thousands of bacterial isolates to identify the unknown microorganism ( [link] ).

A flowchart. A bacterial culture is grown (image is of an agar plate). Then fatty acids are extracted and converted to methyl esters (image is of a test tube). Then gas chromatography analyzes methyl ester fingerprints (image is of a chromatography machine). Then bacteria are identified (image is of a graph). The X axis of the graph is column retention time. The Y axis is of detector signal. The line has various peaks.
Fatty acid methyl ester (FAME) analysis in bacterial identification results in a chromatogram unique to each bacterium. Each peak in the gas chromatogram corresponds to a particular fatty acid methyl ester and its height is proportional to the amount present in the cell. (credit “culture”: modification of work by the Centers for Disease Control and Prevention; credit “graph”: modification of work by Zhang P. and Liu P.)

A related method for microorganism identification is called phospholipid-derived fatty acids (PLFA) analysis . Membranes are mostly composed of phospholipids, which can be saponified (hydrolyzed with alkali) to release the fatty acids. The resulting fatty acid mixture is then subjected to FAME analysis, and the measured lipid profiles can be compared with those of known microorganisms to identify the unknown microorganism.

Bacterial identification can also be based on the proteins produced under specific growth conditions within the human body. These types of identification procedures are called proteomic analysis . To perform proteomic analysis, proteins from the pathogen are first separated by high-pressure liquid chromatography (HPLC) , and the collected fractions are then digested to yield smaller peptide fragments. These peptides are identified by mass spectrometry and compared with those of known microorganisms to identify the unknown microorganism in the original specimen.

Microorganisms can also be identified by the carbohydrates attached to proteins (glycoproteins) in the plasma membrane or cell wall. Antibodies and other carbohydrate-binding proteins can attach to specific carbohydrates on cell surfaces, causing the cells to clump together. Serological tests (e.g., the Lancefield groups tests, which are used for identification of Streptococcus species) are performed to detect the unique carbohydrates located on the surface of the cell.

Resolution

Penny stopped using her new sunscreen and applied the corticosteroid cream to her rash as directed. However, after several days, her rash had not improved and actually seemed to be getting worse. She made a follow-up appointment with her doctor, who observed a bumpy red rash and pus-filled blisters around hair follicles ( [link] ). The rash was especially concentrated in areas that would have been covered by a swimsuit. After some questioning, Penny told the physician that she had recently attended a pool party and spent some time in a hot tub. In light of this new information, the doctor suspected a case of hot tub rash , an infection frequently caused by the bacterium Pseudomonas aeruginosa , an opportunistic pathogen that can thrive in hot tubs and swimming pools, especially when the water is not sufficiently chlorinated. P. aeruginosa is the same bacterium that is associated with infections in the lungs of patients with cystic fibrosis .

The doctor collected a specimen from Penny’s rash to be sent to the clinical microbiology lab. Confirmatory tests were carried out to distinguish P. aeruginosa from enteric pathogens that can also be present in pool and hot-tub water. The test included the production of the blue-green pigment pyocyanin on cetrimide agar and growth at 42 °C. Cetrimide is a selective agent that inhibits the growth of other species of microbial flora and also enhances the production of P. aeruginosa pigments pyocyanin and fluorescein, which are a characteristic blue-green and yellow-green, respectively.

Tests confirmed the presence of P. aeruginosa in Penny’s skin sample, but the doctor decided not to prescribe an antibiotic. Even though P. aeruginosa is a bacterium, Pseudomonas species are generally resistant to many antibiotics. Luckily, skin infections like Penny’s are usually self-limiting; the rash typically lasts about 2 weeks and resolves on its own, with or without medical treatment. The doctor advised Penny to wait it out and keep using the corticosteroid cream. The cream will not kill the P. aeruginosa on Penny’s skin, but it should calm her rash and minimize the itching by suppressing her body’s inflammatory response to the bacteria.

Skin with red raised bumps.
Exposure to Pseudomonas aeruginosa in the water of a pool or hot tub can sometimes cause a skin infection that manifests as “hot tub rash.” (credit: modification of work by “Lsupellmel”/Wikimedia Commons)

Go back to the previous Clinical Focus box.

Key concepts and summary

  • Accurate identification of bacteria is essential in a clinical laboratory for diagnostic and management of epidemics, pandemics, and food poisoning caused by bacterial outbreaks.
  • The phenotypic identification of microorganisms involves using observable traits, including profiles of structural components such as lipids, biosynthetic products such as sugars or amino acids, or storage compounds such as poly-β-hydroxybutyrate.
  • An unknown microbe may be identified from the unique mass spectrum produced when it is analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) .
  • Microbes can be identified by determining their lipid compositions, using fatty acid methyl esters ( FAME ) or phospholipid-derived fatty acids ( PLFA ) analysis .
  • Proteomic analysis , the study of all accumulated proteins of an organism; can also be used for bacterial identification.
  • Glycoproteins in the plasma membrane or cell wall structures can bind to lectins or antibodies and can be used for identification.

Fill in the blank

A FAME analysis involves the conversion of _______ to more volatile _____ for analysis using ____________.

fatty acids, methyl esters, gas chromatography

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True/false

MALDI-TOF relies on obtaining a unique mass spectrum for the bacteria tested and then checking the acquired mass spectrum against the spectrum databases registered in the analysis software to identify the microorganism.

True

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Lancefield group tests can identify microbes using antibodies that specifically bind cell-surface proteins.

False

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Short answer

Compare MALDI-TOF, FAME, and PLFA, and explain how each technique would be used to identify pathogens.

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Questions & Answers

what is biochemistry
Isaac Reply
Biochemistry, sometimes called biological chemistry, is the study of chemical processes within and relating to living organisms. Biochemical processes give rise to the complexity of life. ... The mechanisms by which cells harness energy from their environment via chemical reactions are known as meta
Dejene
what is pathogenic
kelvin Reply
biological pathogen is anything that can produce disease. a pathogen may also be referred to as an infectious agent, or simple a germ.
Dejene
Thanks
kelvin
explain 3 points why gender is relevant to health.
kelvin
wat causes sor throat
Ezeama Reply
Streptococcus
Rajat
Streptococcus
Panda
Gram positive
shamim
streptococcus pyrogenes
Sherma
group A streptococcus pyogenes. But not the only one.
Carlos
does the bac of s.aures gives choronic diseas in bone?
Elyas
causes virus bac group A strep allergies smoking
Elyas
truth also virus
Shania
what z microbiology
Mwelwa Reply
microbiology is all about the scientific study of microorganisms, their life changes, their life span, structural punctuality and their phenomenon.
Ridwan
thanks
micah
Best scope in microbiology
Rahul
wat abut skin
Elyas
whatis scope
Amare
what is scope
Funmi
what happened when someone died and what happened to the blood
Franca Reply
nice question
Attari
when you die you have no memory and the blood dry off
Falere
what microorganisms is all about?
Ridwan
And how many does the microorganisms is classified?
Ridwan
How are microbs organized
Saskia
what is endoparasites and exo parasite and their example
OGEDE Reply
Endo : inside the body Exo/ecto : outside or on the body
Paul
examples please
OGEDE
Louse for exo/ecto Helminths (worms) for endo
Paul
what's prokaryotic
mohaiminul Reply
A prokaryotic cell is a cell with no membrane bounds organelles
Paul
what are the main effect of parasite?
yunusa Reply
nutritional group of micro organism
Mana Reply
what are the charateristic bacteria, eukaryotes, and viruses have in commom
Ami Reply
what type of bacteria ismor serious
Amare
D N A ar purno rup ki
Ruba Reply
বাংলায় দেখবো DNA এর পূর্রন্যরুপ কি
Ruba
what's microbiology
micah
micro biology is the study of micro organisms,those being unicellular multicellular or a cellular
aniisha
deoxyribonucleic acid is a long molecule that contains our unique genetic code.
aniisha
Does the parasite/parasitology are under microbiology or not
yunusa
Define bacteria
Kainat
Bacteria are a type of biological cell & they constitute a large domain of prokaryotic microorganisms.
raisa
what kind of microbiology
Mira
hello what is main function of T and B cells
Ishaq Reply
T cells are involved in cell-mediated immunity while B cells are primarily responsible for humoral immunity (relating to antibodies)
Janet
what is hepatitis B
Sunday
it's viral infection that attacks the liver and can cause both acute and chronic diseases. the virus is most commonly transmitted from mother to child during birth and delivery, as well as through contact with blood or other body fluids...
Dejene
What are fundamental experiment of microbiology?
Lucky Reply
study of microscopic organisms, unicellular and multicellular
Falere
what are the characteristics of bacteria, viruses, and eukaryotes
Ami
bacteria-are living organism. contain a peptidoglycan/lipopolysaccharide cell wall. VIRUS-are considered as organic structure which interact with living organism don't have acell wall. they have many differents like bacteria size -large(1000nm) virus size-smaller(20-400nm). bacteria ribosomes presen
Dejene
virus ribosomes absence. bacteria living organism. virus b/n living and non-living things
Dejene
eukaryotic cell has a true membrane bond nucleus and has other membranous organelles that allow for compartmenalization of function
Dejene
This answer is wrong
Jeffrey Reply
who discovered the growth curve
Joy Reply
Practice MCQ 5

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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