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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 are antibodies?
Happy Reply
they are immune cells that are released by immune system to fight strangers like microbes
Ahmed
a blood protien produce in response to an counteracting a specific antigen
Pooja
Why salmonella typhi is harmful protozoan?
Saily Reply
because make typhoid that is considered as a food born illness
Ahmed
Name any five modes of transmission.
Happy
what are the scientific names and common names of some microbes
Rich Reply
Staphylococcus aureus - staph Streptococcus pyogenes - strep Botulism - Clostridium botulinum Rocky Mountain spotted fever - Rickettsia rickettsii Diphtheria - Corynebacterium diphtheriae Gonorrhea - Neisseria gonorrhoeae
Tammy
what are the limitations of the Koch's postulate
Naa
how a microorganism growth.
Zubair Reply
Is foetus a parasite to his or her mother? if yes and if no ho
Mashauri Reply
y
Yusri
yes
Yashkin
yes
Zubair
yes it a parasite to the mother because it feeds on the mother for survival
Beatrice
yes
Redwan
no
Elasha
definition of a parasite: an organism that lives in or on an organism of ANOTHER species (its host) and benefits by deriving nutrients at the other's expense
Elasha
no because an organism can inky be a parasite if it causes harm to its HOST. And the fetus does not cause harm to its mother under normal conditions
aliyu
yes
Naa
what is biosensor in microbiology
Raja Reply
What are biosensor
Raja
what's are biosensor
Raja
what is microbiology defination
Rinku Reply
microbiology is the study of small or manuit organisms which cannot be seen with our nacked eyes unless with the aid of the microscope
Brandina
is the study of living organisms which are not directly visible to a direct eye but can only be seen under a microscope
Ipa
ok
Ipa
microbiology is the study of living organisms of microscopic size it is also the study of micro organisms with their form structure reproduction psychology metabolism and classification
Priyanka
what are the importances of Microbiology?
Erick Reply
don't know
SINGLE
we obtained the insulin from the bacteria. and some microorganisms are decomposer in ecosystems.
Zubair
nitrogen in the air is fixed into the soil by microbes example is nitrobactor
Matilda
yes
Zubair
how a weak immune region where microorganisms attack easily?
Zubair Reply
with examples differentiate gram positive from gram negative bacteria
Mary Reply
Differentiate gram positive from gram negative
Mary
I have no idea
Zubair
hello
Kuonain
and example of gram negative is E. coli
Pooja
gram positive stain purple when subjected to gram stain whilst gram positive bacterial has thick wall composed of peptidoglycan
Matilda
ok
Zubair
what is a process of gene expression in eukaryotes ?
Nayyab Reply
pls help us with the answer
Abdussalam
spontaneous generation means
Siddhi Reply
hiii
Siddhi
Hello
eman
So, what's next 😂
eman
I have a problem with Micro
eman
I studying things and see question Some thing else
eman
Can u help me, please
eman
What i havta do
eman
firsfall when you studying understand things...
Siddhi
good afternoon dear Friends
yaya
Greeting to everyone in here.
Manka
There is a problem. I need a diagram of a virus with it functions.
Manka
structure of bacterial
Kuyiba
gghhhh
Zubair
group plz i need help in microbio
Kuonain
it really difficult fr me
Kuonain
kashur cha kah
suhail
sorry now I read in class 8th but I can help u
Zubair
Me too. It's very difficult for me
Angela
rod shape
Priyanka
helical
Priyanka
Hello
esike
spherical
Priyanka
peomorphic
Priyanka
I am ryt or not
Priyanka
hi
suhail
hello
DIPTI
Are we together pls what's the topic for the day?
esike
what is innate
Lizzy
innate means natural
Pooja
the genetic makeup of an individual
Matilda
pls i need the common names for the following parasites..ENTEROBIUS VERMICULARIS,NECATOR AMERICANUS,ASCARIES LUMBRICOIDES,TRICHURIS TRICHIURA,TRICHOMONAS VAGINALIS,GIARDIA LAMBLIA,ENTAMOEBA HISTOLYTICA,SCHISTOSOMA MANSONI,SCHISTOSOMAHEMATOBIUM,STROGYLOIDES STERCORALIS,AND TRAPANOSOMA BRUCI GAMBIENSE
timothy
ENTEROBIUS VERMICULARIS and STROGYLOIDES STERCORALIS have the same common name which is pin worm and thread worm
Chinedu
I didn't find the common names for TRICHOMONAS VAGINALIS, GIARDIA LAMBLIA, ENTAMOEBA HISTOLYTICA, TRYPANOSOMA BRUCEI GAMBIENSE
Chinedu
NECATOR AMERICANUS - New world hookworm ASCARIS LUMBRICOIDES- Ascarid TRICHURIS TRICHIURA- Whip worm
Chinedu
Hi guys
Chinedu
Why are myeloblasts not present in the bloodstream?
Chinedu
thank u
Dasaah
is blood from capillary puncture suitable for hormonal assays
NANCY Reply
Direct this question to a hematologist for a better answer please.
LAFIA
In most cases the blood is necessary but it's limited from my own Analysis
Lee
u can do but it depends on your sampling blood its enev or not
Ali
Control indications of cipro, doxicyclin, setracyclin, cloxacyclin, erythromycin
Oslen Reply
oo
babinthe
what is sims position
babinthe 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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