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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

how do i report widal slide results
Isaac Reply
how food can be used as a substrate for the growth of microorganisms
Ashi Reply
what is the importance of understanding chemistry in the field of microbiology and food technology?
Charmaine Reply
What is different between eukaryotes and prokcaryotic
Abia Reply
main difference is that eukaryotic cells possess membrane bound organelles
Iqra
prokaryotes are primitive organisms that doesn't contain membrane bound nucleus or any orgenelle while in eukaryotes membrane bound orgenelles and nucleus is present
Yashi
what s anatomy
jane Reply
its mean body structure, function, and systems
gazi
study of internal structure of living things
Falere
anatomy is the study structure of the made human
isir
anotomy is the actual study of body internally and externally Which include how itis made. for what and what is need of this
Iqra
la science anatomie c'est la science qui nous aide à étudier l'homme
Baraka
okay
Baraka
Ancestor are they real
Rapheal Reply
yes of course
David
yes please
Clark
50 50, depending on the accuracy of the clan records.
Vincent
okay
Baraka
Why protist is not a kingdom of Linnaeus 'S taxonomy?
Neha Reply
I dont know
Jeewraj
Linnaeus used Aristotle's criteria for dividing living organisms into kingdoms. Plants are immobile and insensitive, while animals are mobile and sensitive. the creatures he saw through the microscope were mobile, so he attributed them to animals.
Lad
name the different types of media use in lab to detect the micro organisms
Shehzadi Reply
The different types of media used in the lab to detect the microorganisms is known as cell culture..
Tean
solid media or broth is used to grow and detect microorganisms
Pohor
what is microbiology
Baraka
the study of large living organisms
Pratibha
the study of organisms which are micro in range
Vency
what is autoclaving?
Yashi
process for sterilization
Vency
is a machine used on the process of sterilisation
Clark
what is difference between hot air oven and autoclave as they both are used for sterilization ?
Vency
autoclave basically do moist heat sterilization while hot air oven do sterilization by dry heat.....
Yashi
what is the microbe
HUSSAIN
which method out of these two is best?
Yashi
why human have microbe
HUSSAIN
Hi
Sadam
hi to all
Sadam
depends on what to sterilize
Vency
microbes
Md
what is knowledge
HUSSAIN
well idea
Daniel
what are the importance of microbiologe to a nurse
Enny Reply
it enable a nurse to know the weight and height of a patient
Abotu
it helps the nurse in way as to give first aid to patient on which basis doctor will take diagonos
Iqra
disagree with both of you
Akhtar
ok
Abotu
Your Own idea
Abotu
it helps a nurse to be able to counsel a patient /client
Abotu
cell culture
Tean
Types of micro organisms
Jennifer Reply
Who discovered plant and animal cell
Jennifer
Robert Hooke
Martha
Father of microbiology
Jennifer Reply
Antonie van Leeuwenhoek
Satarupa
Why are mitochondria and chloroplasts unable to multiply outside of a host cell?
Iqra Reply
iqra mitochondria arises from the division of existing mitochondria and they are fused together. they move around inside the cell with the interactions of the cytoskeleton that's why mitochondria unable to multiply outside the host cells chloroplasts has its own separate DNA from the plant cell.
Rana
short not on medical microbiology
Massah Reply
Hy
Iqra
hope you fine
Arif
love you
Arif
Assalamualaikum
Suhaib
arai yeh micro kai sth kis ko love hua
Umer
icrobiology is the scientific study of these microorganisms. Microorganisms are those organisms that are too small to see with the naked eye and include things like bacteria, fungi, and viruses
Suhaib
iqra iqbal
Arif
What is gram syaining?
Arif
to differentiate between gram positive and gram negative bacty
Umer
True
Arif
where you are from
Arif
i m frm sopore..kashmir
Umer
i m frm baramulla .. Kashmir
Umer
and u
Umer
umer
Arif
and i m from afghanistan lovely country
Arif
yes
Umer
thanks
Arif
medical microbiology means study of microorganisms which are beneficial or harmful for the body. mostly which are studied under microscope by structure and shape appearance. e.g bacteria, viruses, bacteriophage, fungus hyphae and some blood and other body fluids parasites e.g plasmodium etc.
Rana
arif bhai gram staining in which we are given different dyes to the bacterial cell wall and cytoplasm and nucleus.e.g giemsa stain
Rana
Gram staining uses crystal violet to stain cell walls iodine as a mordant and a fuchsin or safranin counterstain to mark all bacteria
Umer
Gram staining uses crystal violet to stain cell walls iodine as a mordant and a fuchsin or safranin counterstain to mark all bacteria
Umer
Gram staining uses crystal violet to stain cell walls iodine as a mordant and a fuchsin or safranin counterstain to mark all bacteria
Umer
Omar baba crystal violet iodine 70% alcohol fuchicine or safranine after every step washing must be done
Rana
hello
Cristy
*Fuchsin stain
Cristy
*Safranin
Cristy
thanks
Ganesh
difference between epidermophyton trichophyton and microsporum
Ankita Reply
plz answer the question
Iqra
epidermophyton type of fungi causes superficial and cutaneous mycoses trichophyton is also fungi type including parasitic varieties causes dermatophytosis microsporum is also type of fungi causes dermatophytosis.
Rana
ok
Iqra
Is this app perfect for preparation of exam?
Ankita
Ankita I totally disagree with your respected opinion. it's just minor helpful
Rana
disagree by me ?
Ankita
what is infectious
Fred 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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